Use of substituted 4-biarylbutyric and 5-biarylpentanoic acid derivatives as matrix metalloprotease inhibitors for the treatment of respiratory diseases

ABSTRACT

Novel 4-Biarylbutyric and 5-Biarylpentanoic Acid Derivatives, use of substituted 4-Biarylbutyric and 5-Biarylpentanoic Acid Derivatives as Matrix Metalloprotease Inhibitors for the Treatment of Respiratory Diseases, pharmaceutical compositions containing them, and a process for using them. The compounds of the invention have the generalized formula (I) (T) x A—B—D—E—CO 2 H wherein A is an aryl or heteroaryl rings; B is an aryl or heteroaryl ring or a bond; each T is a substituent group, x is 0, 1, or 2; the group D represents (a), (b), (c), or (d); the group E represents a two or three carbon chain bearing one to three substituent groups which are independent or are involved in ring formation, possible structures being shown in the text and claims; and each of the substituents on E is an independent substituent; and include pharmaceutically acceptable salts thereof.

FIELD

This invention relates to the use of enzyme inhibitors, and moreparticularly, to new and known matrix metalloprotease-inhibiting4-Biarylbutyric Acids and 5-Biarylpentanoic Acids and derivativesthereof, for the prevention and treatment of respiratory diseases.

BACKGROUND

Substituted 4-Biarylbutyric and 5-Biarylpentanoic Acid Derivatives asMatrix Metalloprotease Inhibitors are described in WO 96/15096, WO97/43237, WO 97/43238, WO 97/43239, WO 97/43240, WO 97/43245, WO97/43247 and WO 98/22436.

The matrix metalloproteases (matrix metalloendo-proteinases or MMPs) area family of zinc endoproteinases which include, but are not limited to,interstitial collagenase (MMP-1), stromelysin (proteoglycanase, tansin,or MMP-3), gelatinase A (72 kDa-gelatinase or MMP-2), neutrophilcollagenase (MMP-8), gelatinase B (95 kDa-gelatinase or MMP-9),macrophage elastase (MMP-12) and human collagenase 3 (MMP 13). TheseMMPs are secreted by a variety of cells including fibroblasts,chondrocytes, granulocytes and macrophages along with naturalproteinatious inhibitors known as TIMPs (Tissue Inhibitor ofMetalloProteinase).

All of these MMPs are capable of destroying a variety of connectivetissue components of articular cartilage or basement membranes and awide variety of extracellular matrix proteins. Each MMP is secreted asan inactive proenzyme which must be cleaved in a subsequent step beforeit is able to exert its own proteolytic activity. In addition to thematrix destroying effect, certain of these MMPs such as MMP-3 have beenimplemented as the in vivo activator for other MMPs such as MMP-1 andMMP-9 (A. Ho, H. Nagase, Arch. Biochem. Biophys., 267, 211-16 (1988); Y.Ogata, J. J. Enghild, H. Nagase, J. Biol. Chem., 267, 3581-84 (1992)).Thus, a cascade of proteolytic activity can be initiated by an excess ofMMP-3. It follows that specific MMP-3 inhibitors should limit theactivity of other MMPs that are not directly inhibited by suchinhibitors.

In addition to its ability to degrade extracellular matrix proteinsMMP-12 has also been shown to hydrolyse elastin (R. P. Mecham, T. J.Broekelmann, C. J. Fliszar, S. D. Shapiro, H. G. Welgus, R. M. Senior,J. Biol. Chem., 272, 18071-18076 (1997)). This activity is shared byother MMP enzymes, specifically MMP-2 and MMP-9.

It has also been reported that MMP-3, MMP-9 and MMP-12 can cleave andthereby inactivate the endogenous inhibitors of other proteinases suchas elastase (P. G. Winyard, Z. Zhang, K. Chidwick, D. R. Blake, R. W.Carrell, G. Murphy, FEBS Letts., 279, 1, 91-94 (1991); T. J. Gronski, R.L. Martin, D. K. Kobayashi, B. C. Walsh, M. C. Holman, M. Huber, H. E.Van Wart, S. D. Shapiro, J. Biol. Chem., 272, 12189-12194 (1997)).Inhibitors of these MMP enzymes could thus influence the activity ofother destructive proteinases by modifying the level of their endogenousinhibitors.

Macrophages from MMP-12 knock-out mice have a diminished capacity todegrade extracellular matrix components and to penetrate reconstitutedbasement membranes both in vitro and in vivo (J. M. Shipley, R. L.Wesselschmidt, D. K. Kobayashi, T. J. Ley, S. D. Shapiro, PNAS, 93,3942-3946 (1996)). These results support the hypothesis that MMP-12 isrequired for macrophage mediated extracellular proteolysis and tissueinvasion. In addition, MMP-12 knock-out mice do not develop emphysema orshow elevated macrophage levels in response to smoking, whereas wildtype mice do (R. D. Hautamaki, D. K. Kobayashi R. M. Senior, S. D.Shapiro, Science, 277, 2002-2004 (1997)). Therefore there is strongevidence supporting a role of M-12, secreted by activated alveolarmacrophages, in the development of pulmonary emphysema In patients withemphysema and smoking subjects both MMP-1, -8, -9 and -12 released fromalveolar macrophages and neutrophils are also implicated in thepathogenesis of COPD.

By means of their proteolytic activity, matrix metallo-proteases areinvolved in a number of respiratory diseases, e.g. the following:

-   -   asthma; chronic obstructive pulmonary disease including chronic        bronchitis and emphysema; cystic fibrosis; bronchiectasis; adult        respiratory distress syndrom (ARDS); allergic respiratory        disease including allergic rhinitis; diseases linked to TNF_(α)        production including acute pulmonary fibrotic diseases,        pulmonary sarcoidosis, silicosis, coal worker's pneumoconiosis,        alveolar injury.

Evidence for the involvement of matrix metalloproteases in variousrespiratory diseases is provided by the following references:

a) COPD, Finlay et al. Thorax 1997, 52, 502 chronic Am. J. Resp. CritCare Med. 1997, bronchitis 156, 240 and Cateldo et al. Am. J. Resp.Crit. Care Med. 1998, emphysema 157, A 502 Sedura et al. Am. J. Resp.Crit. Care Med. 1998, 157, A 568 Shapiro et al. J. Biol. Chem. 1993,268, 23824 Kostan et al. Am. J. Resp. Crit. Care Med. 1998, 157, A 143Riccobono Eur. Resp. J. 1997, 10, 26S b) bronchi- Sepper et al. Chest1995, 107, 1641 ectasis Sepper et al. Eur. Resp. J. 1997, 10, 278S c)cystic Delacourt et al. Am. J. Resp. Crit. Care Med. 1995, fibrosis 152,A 764 Power et al. Am. J. Resp. Crit. Care Med. 1994, 150, 818 d) asthmaShute et al. Int. Arch. Allergy Immunol. 1997, 111 Ohno et al. Am. J.Resp. Cell. Mol. Biol. 1997, 16, 212 Mantino et al. Am. J. Resp. Cell.Mol. Biol. 1997, 17, 583 Okada et al. Am. J. Resp. Cell. Mol. Biol.1997, 17, 519 e) ARDS Delclaux et al. Am. J. Physiol. 1997, 272, L442.

In addition to chronic lung diseases a number of other conditions arethought to be mediated by excess or undesired matrix-destroyingmetalloprotease activity or by an imbalance in the ratio of the MMPs tothe TIMPs or through the action of the release of TNF.

MMP inhibitors may also be useful in the inhibition of other mammalianmetalloproteases such as the adamalysin family (or ADAMs) whose membersinclude TNF_(α) converting enzyme (TACE) and ADAM-10, which can causethe release of TNF from cells.

SUMMARY

This invention relates to the use for the prevention and treatment ofrespiratory diseases of new and known compounds having matrixmetallprotease inhibitory activity of the generalized formula (I):(T)_(x)A—B—D—E—CO₂H.  (I)In the above generalized formula (I), (T)_(x)A represents a substitutedor unsubstituted aromatic 6-membered ring or heteroaromatic 5-6 memberedring containing 1-2 atoms of N, O, or S. T represents one or moresubstituent groups, the subscript x represents the number of suchsubstituent groups, and A represents the aromatic or heteroaromaticring, designated as the A ring or A unit. When N is employed inconjunction with either S or O in the A ring, these heteroatoms areseparated by at least one carbon atom.

The substituent group(s) T are independently selected from the groupconsisting of halogen; alkyl; haloalkyl; haloalkoxy; alkenyl; alkynyl;—(CH₂)_(p)Q in which p is 0 or an integer of 1-4; -alkenyl-Q in whichthe alkenyl moiety comprises 2-4 carbons; and alkynyl-Q in which thealkynyl moiety comprises 2-7 carbons. Q in the latter three groups isselected from the group consisting of aryl, heteroaryl, —CN, —CHO, —NO₂,—CO₂R², —OCOR², —SOR³, —SO₂R³, —CON(R⁴)₂, —SO₂N(R⁴)₂, —COR², —N(R⁴)₂,—N(R²)COR², —N(R²)CO₂R³, —N(R²)CON(R⁴)₂, —CHN₄, OR⁴, and —SR⁴.

In these formulae R² represents H, alkyl, aryl, heteroaryl, arylalkyl,or heteroaryl-alkyl. R³ represents alkyl, aryl, heteroaryl, arylalkyl,or heteroaryl-alkyl. R⁴ represents H; alkyl; aryl; heteroaryl;arylalkyl; heteroaryl-alkyl; alkenyl; alkynyl; alkyleneoxy,polyalkyleneoxy, alkylenethio or alkyleneamino terminated with H, alkyl,or phenyl; haloalkyl; lower alkoxycarbonyl; or acyl. When two R⁴ groupsare situated on a nitrogen, they may be joined by a bond to form aheterocycle, such as, for example, a morpholine, thiomorpholine,pyrrolidine, or piperidine ring.

Unsaturation in a moiety which is attached to Q or which is part of Q isseparated from any N, O, or S of Q by at least one carbon atom. The Aring may be unsubstituted or may carry up to 2 substituents T.Accordingly, the subscript x is 0, 1, or 2.

In the generalized formula (I), B represents a bond or an optionallysubstituted aromatic 6-membered ring or a heteroaromatic 5-6 memberedring containing 1-2 atoms of N, O, or S. When B is a ring, it isreferred to as the B ring or B unit. When N is employed in conjunctionwith either S or O in the B ring, these heteroatoms are separated by atleast one carbon atom. There may be 0-2 substituents T on ring B.

In the generalized formula (I), D represents

in which R² is defined as above and each R² may be the same ordifferent.

In the generalized formula (I), E represents a chain of n carbon atomsbearing m substituents R⁶, in which the R⁶ groups are independentsubstituents, or constitute spiro or nonspiro rings. Rings may be formedin two ways: a) two groups R⁶ are joined, and taken together with thechain atom(s) to which the two R⁶ group(s) are attached, and anyintervening chain atoms, constitute a 3-7 membered ring, or b) one groupR⁶ is joined to the chain on which this one group R⁶ resides, and takentogether with the chain atom(s) to which the R⁶ group is attached, andany intervening chain atoms, constitutes a 3-7 membered ring. The numbern of carbon atoms in the chain is 2 to 4, and the number m of R⁶substituents is an integer of 1-3.

Each group R⁶ is independently selected from the group consisting of:

-   -   fluorine;    -   hydroxyl, with the proviso that a single carbon atom may bear no        more than one hydroxyl group;    -   alkyl;    -   aryl;    -   heteroaryl;    -   arylalkyl;    -   heteroaryl-alkyl;    -   alkenyl;    -   aryl-substituted alkenyl;    -   heteroaryl-substituted alkenyl;    -   alkynyl;    -   aryl-substituted alkynyl;    -   heteroaryl-substituted alkynyl;    -   —(CH₂)_(t)R⁷, wherein t is 0 or an integer of 1-5 and        -   R⁷ is selected from the group consisting of:            -   N-phthalinidoyl;            -   N-(1,2-naphthalenedicarboximidoyl);            -   N-(2,3-naphthalenedicarboximidoyl);            -   N-(1,8-naphthalenedicarboximidoyl);            -   N-indoloyl;            -   N-(2-pyrrolodinonyl);            -   N-succiniimidoyl;            -   N-maleimidoyl;            -   3-hydantoinyl;            -   1,2,4-urazolyl;            -   amido;            -   urethane;            -   urea;            -   nonaromatic substituted or unsubstituted heterocycles                containing and connected through a N atom, and                comprising one or two additional N, O, S, SO, or SO₂,                and containing zero, one or two carbonyls, and                optionally bearing a fused benzene or pyridine ring;            -   amino;            -   corresponding heteroaryl moieties in which the aryl                portion of an aryl-containing R⁷ group comprises 4-9                carbons and at least one N, O, or S heteroatom;    -   (CH₂)_(v)ZR⁸ in which v is 0 or an integer of 1-4, wherein        -   Z represents        -    and        -   R⁸ is selected from the group consisting of:            -   alkyl;            -   aryl;            -   heteroaryl;            -   arylalkyl;            -   heteroaryl-alkyl; and            -   —C(O)R⁹ in which R⁹ represents alkyl of at least two                carbons, aryl, heteroaryl, arylalkyl, or                heteroaryl-alkyl;        -   and with the further provisos that            -   when R⁸ is —C(O)R⁹, Z is S or O;            -   when Z is O, R⁸ may also be alkyleneoxy or                polyalkyleneoxy terminated with H, alkyl, or phenyl; and    -   trialkylsilyl-substituted alkyl.

Furthermore, aryl or heteroaryl portions of any of the T or R⁶ groupsoptionally may bear up to two substituents selected from the groupconsisting of —(CH₂)_(y)C(R⁴)(R³)OH, —(CH₂)_(y)OR⁴, —(CH₂)_(y)SR⁴,—(CH₂)_(y)S(O)R⁴, —(CH₂)_(y)S(O)₂R⁴, —(CH₂)_(y)SO₂N(R⁴)₂,—(CH₂)_(y)N(R⁴)₂, —(CH₂)_(y)N(R⁴)COR¹², —OC(R⁴)₂O— in which both oxygenatoms are connected to the aryl ring, (CH₂)_(y)COR⁴, —(CH₂)_(y)CON(R⁴)₂,—(CH₂)_(y)CO₂R⁴, —(CH₂)_(y)OCOR⁴, -halogen, —CHO, —CF₃, —NO₂, —CN, and—R³, in which y is 0-4. R³ and R⁴ are defined as above; in addition, anytwo R⁴ which are attached to one nitrogen may be joined to form aheterocycle such as morpholine, thiomorpholine, pyrrolidine, or apiperidine ring.

Pharmaceutically acceptable salts of these compounds as well as commonlyused prodrugs of these compounds such as O-acyl derivatives of inventioncompounds which contain hydroxy groups are also within the scope of theinvention.

In most related reference compounds of the prior art, the biphenylportion of the molecule is unsubstituted, and the propanoic or butanoicacid portion is either unsubstituted or has a single methyl or phenylgroup. Presence of the larger phenyl group has been reported to causeprior art compounds to be inactive as anti-inflammatory analgesicagents. See, for example, R. G. Child, et al., J. Pharm. Sci., 66,466-476 (1977). By contrast, it has now been found that compounds whichexhibit potent MMP inhibitory activity contain a substituent ofsignificant size on the a propanoic or butanoic portion of the molecule.The biphenyl portions of the best MMP inhibitors also preferably containa substituent on the 4′ position, although when the propanoic orbutanoic portions are optimally substituted, the unsubstituted biphenylcompounds of the invention have sufficient activity to be consideredrealistic drug candidates.

The compounds of the present invention exhibit good activity for MMP-2,MMP-3, MMP-8, MMP-9, MMP-12 and MMP-13, and a good selectivity for theseMMP's over other MMP's such as MMP-1 and MMP-7.

In addition to the above-described compounds, the invention also relatesto pharmaceutical compositions having matrix metalloprotease inhibitoryactivity, which compositions comprise a compound of the invention asdescribed above and in more detail in the detailed description below,and a pharmaceutically acceptable carrier.

As a result of the abovementioned selectivity profile, the compounds ofthe present invention are especially suitable for the treatment ofrespiratory diseases.

Therefore, the invention also relates to a method of treating a mammalsuch as a human, a farm animal, or a domestic pet, to achieve an effect,in which the effect is: treatment and prevention of asthma; chronicobstructive pulmonary disease including chronic bronchitis andemphysema; cystic fibrosis; bronchiectasis; adult respiratory distresssyndrome (ARDS); allergic respiratory disease including allergicrhinitis; diseases linked to TNF_(α) production including acutepulmonary fibrotic diseases, pulmonary sarcoidosis, silicosis, coalworker's pneumoconiosis, alveolar injury; the method comprisingadministering an amount of a compound of the invention as describedabove, and in more detail in the detailed description below, which iseffective to inhibit the activity of at least one matrixmetalloprotease, resulting in achievement of the desired effect.

DETAILED DESCRIPTION

More particularly preferred are for the use for the prevention andtreatment of respiratory diseases are compounds having matrixmetalloprotease inhibitory activity of the generalized formula:(T)_(x)A—B—D—E—CO₂H  (I)in which (T)_(x)A represents a substituted or unsubstituted aromatic orheteroaromatic moiety selected from the group consisting of:

in which R¹ represents H or alkyl of 1-3 carbons.

In these structures, the aromatic ring is referred to as the A ring or Aunit, and each T represents a substituent group, referred to as a Tgroup or T unit. Substituent groups T are independently selected fromthe group consisting of: the halogens —F, —Cl, —Br, and —I; alkyl of1-10 carbons; haloalkyl of 1-10 carbons; haloalkoxy of 1-10 carbons;alkenyl of 2-10 carbons; alkynyl of 2-10 carbons; —(CH₂)_(p)Q in which pis 0 or an integer 1-4; -alkenyl-Q in which the alkenyl moiety comprises2-4 carbons; and -alkynyl-Q in which the alkenyl moiety comprises 2-7carbons. Q in each of the latter three groups is selected from the groupconsisting of aryl of 6-10 carbons; heteroaryl comprising 4-9 carbonsand at least one N, O, or S heteroatom; —CN; —CHO; —NO₂; —CO₂R²; —OCOR²;—SOR³; —SO₂R³; CON(R⁴)₂; —SO₂N(R⁴)₂; —C(O)R²; —N(R⁴)₂; —N(R²)COR²;—N(R²)CO₂R³; —N(R²)CON(R⁴)₂; —CHN₄; —OR⁴; and —SR⁴. The groups R², R³,and R⁴ are defined as follows.

-   -   R² represents H; alkyl of 1-6 carbons; aryl of 6-10 carbons;        heteroaryl comprising 4-9 carbons and at least one N, O, or S        heteroatom; arylalkyl in which the aryl portion contains 6-10        carbons and the alkyl portion contains 1-4 carbons; or        heteroaryl-alkyl in which the heteroaryl portion comprises 4-9        carbons and at least one N, O, or S heteroatom and the alkyl        portion contains 1-4 carbons.    -   R³ represents alkyl of 1-4 carbons; aryl of 6-10 carbons;        heteroaryl comprising 4-9 carbons and at least one N, O, or S        heteroatom; arylalkyl in which the aryl portion contains 6-10        carbons and the alkyl portion contains 1-4 carbons, or        heteroaryl-alkyl in which the heteroaryl portion comprises 4-9        carbons and at least one N, O, or S heteroatom and the alkyl        portion contains 1-4 carbons,    -   R⁴ represents H; alkyl of 1-12 carbons; aryl of 6-10 carbons;        heteroaryl comprising 4-9 carbons and at least one N, O, or S        heteroatom; arylalkyl in which the aryl portion contains 6-10        carbons and the alkyl portion contains 1-4 carbons;        heteroaryl-alkyl in which the heteroaryl portion comprises 4-9        carbons and at least one N, O, or S heteroatom and the alkyl        portion contains 1-4 carbons; alkenyl of 2-12 carbons; alkynyl        of 2-12 carbons; —(C_(q)H_(2q)O)_(r)R⁵ in which q is 1-3, r is        1-3, and R⁵ is H provided q is greater than 1, or R⁵ is alkyl of        1-4 carbons, or phenyl; alkylenethio terminated with H, alkyl of        1-4 carbons, or phenyl; alkyleneamino terminated with H, alkyl        of 1-4 carbons, or phenyl; —(CH₂)_(s)X in which s is 1-3 and X        is halogen; —C(O)OR²; or —C(O)R².

When two R⁴ groups are situated on a nitrogen, they may be joined by abond to form a heterocycle, such as, for example, a morpholine,thiomorpholine, pyrrolidine, or piperidine ring.

Any unsaturation in a moiety which is attached to Q or which is part ofQ is separated from any N, O, or S of Q by at least one carbon atom, andthe number of substituents, designated x, is 0, 1, or 2.

In the generalized formula (I), B represents a bond or an optionallysubstituted aromatic or heteroaromatic ring selected from the groupconsisting of:

in which R¹ is defined as above. These rings are referred to as the Bring or B unit. There may be 0-2 substituents T on the B ring, T beingdefined as above.

In the generalized formula (I), D represents the moieties

in which R² is defined as above and each R² may be the same ordifferent.

In the generalized formula (I), E represents a chain of n carbon atomsbearing m substituents R⁶, referred to as R⁶ groups or R⁶ units. The R⁶groups are independent substituents, or constitute spiro or nonspirorings. Rings may be formed in two ways: a) two groups R⁶ are joined, andtaken together with the chain atom(s) to which the two R6 group(s) areattached, and any intervening chain atoms, constitute a 3-7 memberedring, or b) one group R⁶ is joined to the chain on which this one groupR⁶ resides, and taken together with the chain atom(s) to which the R⁶group is attached, and any intervening chain atoms, constitutes a 3-7membered ring. The number n of carbon atoms in the chain is 2 or 3, andthe number m of R⁶ substituents is an integer of 1-3.

Each group R⁶ is independently selected from the group consisting of thesubstituents listed below as items 1)-16).

-   -   1) fluorine;    -   2) hydroxyl, with the proviso that a single carbon atom may bear        no more than one hydroxyl group;    -   3) alkyl of 1-10 carbons;    -   4) aryl of 6-10 carbons;    -   5) heteroaryl comprising 4-9 carbons and at least one N, O, or S        heteroatom;    -   6) arylalkyl in which the aryl portion contains 6-10 carbons and        the alkyl portion contains 1-8 carbons;    -   7) heteroaryl-alkyl in which the heteroaryl portion comprises        4-9 carbons and at least one N, O, or S heteroatom, and the        alkyl portion contains 1-8 carbons;    -   8) alkenyl of 2-10 carbons;    -   9) aryl-alkenyl in which the aryl portion contains 6-10 carbons        and the alkenyl portion contains 2-5 carbons;    -   10) heteroaryl-alkenyl in which the heteroaryl portion comprises        4-9 carbons and at least one N, O, or S heteroatom and the        alkenyl portion contains 2-5 carbons;    -   11) alkynyl of 2-10 carbons;    -   12) aryl-alkynyl in which the aryl portion contains 6-10 carbons        and the alkynyl portion contains 2-5 carbons;    -   13) heteroaryl-alkynyl in which the heteroaryl portion comprises        4-9 carbons and at least one N, O, or S heteroatom and the        alkynyl portion contains 2-5 carbons;    -   14) —(CH₂)_(t)R⁷ in which t is 0 or an integer of 1-5 and R⁷ is        selected from the group consisting of        as well as corresponding heteroaryl moieties in which the aryl        portion of an aryl-containing R⁷ group comprises 4-9 carbons and        at least one N, O, or S heteroatom. In such R⁷ groups, Y        represents O or S; R¹, R², and R³ are as defined above; and u is        0, 1, or 2;    -   15) —(CH₂)_(v)ZR⁸ in which v is 0 or an integer of 1 to 4; Z        represents —S—, —S(O)—, —SO₂—, —O—, carbonyl, or —CH(OH)—; and        R⁸ is selected from the group consisting of: alkyl of 1 to 12        carbons; aryl of 6 to 10 carbons; heteroaryl comprising 4-9        carbons and at least one N, O, or S heteroatom; arylalkyl in        which the aryl portion contains 6 to 12 carbons and the alkyl        portion contains 1 to 4 carbons; heteroaryl-alkyl in which the        aryl portion comprises 4-9 carbons and at least one N, O, or S        heteroatom and the alkyl portion contains 1-4 carbons; —C(O)R⁹        in which R⁹ represents alkyl of 2-6 carbons, aryl of 6-10        carbons, heteroaryl comprising 4-9 carbons and at least one N,        O, or S heteroatom, or arylalkyl in which the aryl portion        contains 6-10 carbons or is heteroaryl comprising 4-9 carbons        and at least one N, O, or S heteroatom, and the alkyl portion        contains 1-4 carbons, with the provisos that        -   when R⁸ is —C(O)R⁹, Z is —S— or —O—;        -   when Z is —O—, R⁸ may also be —(C_(q)H_(2q)O)_(r)R⁵ in which            q, r, and R⁵ are as defined above;    -   16) —(CH₂)_(w)Si(R¹⁰)₃ in which w is an integer of 1 to 3, and        R¹⁰ represents alkyl of 1 to 4 carbons.

In addition, aryl or heteroaryl portions of any of the T or R⁶ groupsoptionally may bear up to two substituents selected from the groupconsisting of —(CH₂)_(y)C(R⁴)(R³)OH, —(CH₂)_(y)OR⁴), —(CH₂)_(y)SR⁴),—(CH₂)_(y)S(O)R⁴), —(CH₂)_(y)S(O)₂R⁴), —(CH₂)_(y)SO₂N(R⁴))₂,—(CH₂)_(y)N(R⁴))₂, —(CH₂)_(y)N(R⁴))COR³, —OC(R⁴))₂O— in which bothoxygen atoms are connected to the aryl ring, —(CH₂)_(y)COR⁴,—(CH₂)_(y)CON(R⁴))₂, —(CH₂)_(y)CO₂R⁴), —(CH₂)_(y)OCOR⁴), -halogen, —CHO,—CF₃, —NO₂, —CN, and —R³, in which y is 0-4; R³ is defined as above; R⁴is defined as above and in addition, any two R⁴ which are attached toone nitrogen may be joined to form a heterocycle, such as a morpholine,thiomorpholine, pyrrolidine, or piperidine ring.

Pharmaceutically acceptable salts of these compounds as well as commonlyused prodrugs of these compounds such as O-acyl derivatives of thesecompounds are also within the scope of the invention.

In the compounds of the invention, the following are preferred.

The substituent group T, when it is on the ring A, is preferablyhalogen, 1-alkynyl-Q, or an ether OR⁴ wherein R⁴ is preferably alkyl of1-12 carbons or arylalkyl in which the aryl portion is 6-10 carbons andthe alkyl portion contains 1-4 carbons. Most preferably, T is halogen,or —C═C—(CH₂)_(t)OH in which t is an integer of 1-5, and when T is OR⁴,R⁴ is alkyl of 1-6 carbons, or benzyl.

The subscript x, which defines the number of T substituents, ispreferably 1 or 2, most preferably 1, and this substituent T ispreferably on the 4-position of ring A.

The A ring is preferably a phenyl or thiophene ring, most preferablyphenyl. The A ring preferably bears at least one substituent group T,preferably located on the position furthest from the position of the Aring which is connected to the B ring.

The B moiety of generalized formula (I) is a bond or a substituted orunsubstituted aromatic or heteroaromatic ring, in which any substituentsare groups which do not cause the molecule to fail to fit the activesite of the target enzyme, or disrupt the relative conformations of theA and B rings, such that they would be detrimental. Such groups may be,but are not limited to, moieties such as lower alkyl, lower alkoxy, CN,NO₂, halogen, etc. The B moiety is preferably a 1,4-phenylene or2,5-thiophene ring, most preferably 1,4-phenylene.

The D unit is most preferably a carbonyl or a —CHOH— group.

The group R⁶ is preferably:

-   -   1) arylalkyl wherein the aryl portion contains 6-10 carbons and        the alkyl portion contains 1-8 carbons;    -   2) —(CH₂)_(t)R⁷ wherein t is 0 or an integer of 1-5 and R⁷ is an        imidoyl group fused to an aromatic residue, or the        1,2,3-benzotriazin-4(3H)-one-3-yl group; or    -   3) —(CH₂)_(v)ZR⁸ wherein v is 0 or an integer of 1-4, Z is S or        O, and R⁸ is aryl of 6-10 carbons or arylalkyl wherein the aryl        portion contains 6 to 12 carbons and the alkyl portion contains        1 to 4 carbons.

The group R⁶ is most preferably one of the following, and in these, anyaromatic moiety is preferably substituted:

-   -   1) arylalkyl wherein the aryl portion is phenyl and the alkyl        portion contains 1-4 carbons;    -   2) —(CH₂)_(t)R⁷ wherein t is an integer of 1-3, and R⁷ is        N-phthalimidoyl, 1,2,3-benzotriazin-4(3H)-one-3-yl,        N-(1,2-naphthalenedicarboximidoyl),        N-(2,3-naphthalenedicarboximidoyl), or        N-(1,8-naphthalenedicarboximidoyl); or    -   3) —(CH₂)_(v)ZR⁸ wherein v is an integer of 1-3, Z is S, and R⁸        is phenyl.

It is to be understood that as used herein, the term “alkyl” meansstraight, branched, cyclic, and polycyclic materials. The term“haloalkyl” means partially or fully halogenated alkyl groups such as—(CH₂)₂Cl, —CF₃ and —C₆F₁₃, for example.

In one of its embodiments, the invention relates to compounds ofgeneralized formula (I) in which at least one of the units A, B, T, andR⁶ comprises a hetero-aromatic ring. Preferred heteroaromaticring-containing compounds are those in which the heteroaryl groups areheteroaryl of 4-9 carbons comprising a 5-6 membered heteroaromatic ringcontaining O, S; or NR¹ when the ring is 5-membered, and N when saidring is 6-membered. Particularly preferred hetero-aromaticring-containing compounds are those in which at least one of the A and Bunits comprises a thiophene ring. When A unit is thiophene, it ispreferably connected to B unit at position 2 and carries one substituentgroup T on position 5. When B Unit is thiophene, it is preferablyconnected through positions 2 and 5 to D and A units respectively.

In another embodiment, the invention relates to compounds of generalizedformula (I), in the E unit of which n is 2 and m is 1. These compoundsthus possess two carbon atoms between the D unit and carboxyl group, andcarry one substituent on this two carbon chain.

In another of its embodiments, the invention relates to compounds ofgeneralized formula (I) in which the A ring is a substituted orunsubstituted phenyl group, the B ring is phenylene, and aryl portionsof any aryl containing T and R⁶ moieties contain only carbon in therings. These compounds thus contain no heteroaromatic rings.

In another of its embodiments, the invention relates to compounds ofgeneralized formula (I) in which m is 1 and R⁶ is an independentsubstituent. These compounds are materials which contain only a singlesubstituent R⁶ on the E unit, and this substituent in not involved in aring.

Preferred compounds of general formula (I) in which R⁶ is —(CH₂)_(t)R⁷have t as an integer of 1-5. Preferred compounds of general formula (I)in which R⁶ is —(CH₂)_(v)ZR⁸ have v as an integer of 1-4 and Z as —S— or—O—. Preferred compounds of general formula (I) in which R⁶ is alkylcontain 4 or more carbons in said alkyl and those in which R⁶ isarylalkyl contain 2-3 carbons in the alkyl portion of said arylalkyl.

In another of its embodiments, the invention relates to compounds ofgeneralized formula (I) in which the number of substituents m on the Eunit is 2 or 3; and when m is 2, both groups R⁶ are independentsubstituents, or together constitute a spiro ring, or one group R⁶ is anindependent substituent and the other constitutes a spiro ring; and whenm is 3, two groups R⁶ are independent substituents and one group R⁶constitutes a ring, or two groups R⁶ constitute a ring and one group R⁶is an independent substituent, or three groups R⁶ are independentsubstituents. This subset therefore contains compounds in which the Eunit is di- or trisubstituted, and in the disubstituted case any ringsformed by one or both R⁶ groups are spiro rings, and in thetrisubstituted case, the R⁶ groups may form either spiro or nonspirorings.

In another of its embodiments, the invention relates to compounds ofgeneralized formula (I) in which the number of substituents m on the Eunit is 1 or 2; and when m is 1, the group R⁶ constitutes a nonspiroring; and when m is 2, both groups R⁶ together constitute a nonspiroring or one group R⁶ is an independent substituent and the otherconstitutes a nonspiro ring. This subset therefore contains compounds inwhich the E unit carries one or two substituents R⁶, and at least one ofthese substituents is involved in a nonspiro ring.

More particularly, representative compounds of generalized formula (I)in which one or more of the substituent groups R⁶ are involved information of nonspiro rings have E units of the following structures:

in which a is 0, 1, or 2; b is 0 or 1; c is 0 or 1; d is 0 or 1; c+d is0 or 1; e is 1-5; f is 1-4; g is 3-5; h is 2-4; i is 0-4; j is 0-3; k is0-2; the total number of groups R⁶ is 0, 1, or 2; U represents O, S, orNR¹; and z is 1 or 2; Each group R¹⁴ is independently selected from thegroup consisting of: alkyl of 1-9 carbons; arylalkyl in which the alkylportion contains 1-7 carbons and the aryl portion contains 6-10 carbons;alkenyl of 2-9 carbons; aryl-substituted alkenyl in which the alkenylportion contains 2-4 carbons and the aryl portion contains 6-10 carbons;alkynyl of 2-9 carbons; aryl-substituted alkynyl in which the alkynylportion contains 2-4 carbons and the aryl portion contains 6-10 carbons;aryl of 6-10 carbons; —COR²; —CH(OH)R²; —CO₂R³; —CON(R²)₂; —(CH₂)_(t)R⁷in which t is 0 or an integer of 1-4; and —(CH₂)_(v)ZR⁸ in which v is 0or an integer of 1 to 3, and Z represents —S—, S(O), SO₂, or —O—. R¹,R⁷, and R⁸ have been defined above.

Other preferred compounds of generalized formula (I) in which one ormore of the substituent groups R⁶ are involved in formation of nonspirorings have E units of the following structures:

in which a, b, c, d, (c+d), e, g, i, k, the total number of groups R⁶,U, and R¹⁴ are as defined above.

Other preferred compounds for the use for the prevention and treatmentof respiratory diseases of generalized formula (I), in which one or moreof the substituent groups R⁶ are involved in formation of nonspiro ringshave the formula

in which the subscript x is 1 or 2; one substituent T is located on the4-position of the A ring, relative to the point of attachment betweenthe A and B rings; e is 2 or 3; and R¹⁴ is as defined above.

More preferred compounds are those shown in table 1:

TABLE 1 No. Structure C-I

C-II

C-III

C-IV

C-V

C-VI

C-VII

C-VIII

C-IX

C-X

C-XI

C-XII

C-XIII

C-XIV

C-XV

C-XVI

C-XVII

C-XVIII

C-XIX

C-XX

C-XXI

C-XXII

C-XXIII

C-XXIV

C-XXV

C-XXVI

C-XXVII

C-XXVIII

C-XXIX

C-XXX

C-XXXI

C-XXXII

C-XXXIII

C-XXXIV

C-XXXV

C-XXXVI

C-XXXVII

C-XXXVIII

C-XXXIX

In the above structures, the term “racemate” in case of the cyclopentanederivatives refers to the trans, trans-diastereomer, that is, e.g. forexamples C-VII, C-XI, C-XVI to C-XVIII and C-XXIX to C-XXXV a 1S/R,2S/R, 5R/S relationship.

Especially preferred is the use of the following compound:(+)-4-(4′-chloro[1,1′-biphenyl]-4-yl)-2-[2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl]-4-oxobutanoicacid

In another aspect of the invention the following new compounds of thegeneral formula (I′)

are provided, wherein CO—E—CO₂H represents a3-carboxyl-5-R⁷-pentan-1-on-1-yl-residue and the substituents T and R⁷have the meaning indicated in the following table:

TABLE 2 racemate, (+)- or (−)- T R¹ entantiomer OEt

(+) ; OEt

(−) ; OAc

rac ; OH

rac ; Cl

rac ; Br

(+) ; Br

(−) ; Cl

(+) ; Cl

(−) ; CN

rac or OCF₃

rac .

Especially preferred is the following compound:(+)-2-[2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl-4-(4′-ethoxy[1,1′-biphenyl]-4-yl)-4-oxobutanicacid

In another aspect of the invention, use of compounds of the generalformula (I′)

wherein

-   -   T is (C₁-C₄)-alkoxy, chloride, bromide, fluoride, acetoxy,        hydroxy, cyano, trifluoromethyl or trifluoromethoxy,    -   CO—E—CO₂H represents a 3-carboxyl-5-R⁷-pentan-1-on-1-yl- or a        2-carboxyl-3-(R⁷-methyl)-cyclopentan-1-yl)carbonyl-residue, and    -   R⁷ represents a group of the formula        and their salts, is a preferred embodiment.

Especially preferred is the use of the following compound:(+)-2-[2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl]-4-(4′-ethoxy[1,1′-biphenyl]-4-yl)-4-oxobutanoicacid,

General Preparative Methods:

The compounds of the invention may be prepared by use of known chemicalreactions and procedures as described in details in WO 96/15096, WO97/43237, WO 97/43238, WO 97/43239, WO 97/43240, WO 97/43245, WO97/43247and WO 98/22436. Nevertheless, the following general preparative methodsare presented to aid the reader in synthesizing the inhibitors. Generalmethods A through K may be used to prepare appropriately substituted4-biaryl-4-oxobutanoic acids, 4-aryl-4-oxobutanoic acids,5-biaryl-5-oxopentanoic acids, or 5-aryl-5-oxopentanoic acids. Thesegeneral methods are also found in WO 9615096 (23 May, 1996) along withexemplary preparations of the keto acids. The choice of a specificsynthetic method is dictated by the proviso that the conditions used donot effect undesired changes in the T or R⁶ moieties of the compoundsprepared.

All variable groups of these methods are as described in the genericdescription if they are not specifically defined below. The variablesubscript n is independently defined for each method. When a variablegroup with a given symbol (i.e. R⁶ or T) is used more than once in agiven structure, it is to be understood that each of these groups may beindependently varied within the range of definitions for that symbol. Asdefined above, the compounds of the invention contain as the E unit achain of 2 or 3 carbon atoms bearing 1 to 3 substituents R⁶ which arenot defined as H. By contrast, it is to be noted that in the generalmethod schemes below, the R⁶ groups are used as if their definitionincludes H, to show where such R⁶ groups may exist in the structures,and for ease in drawing. No change in the definition of R⁶ is intendedby this non-standard usage, however. Thus, only for purposes of thegeneral method schemes below, R⁶ may be H in addition to the moietiesset forth in the definition of R⁶. The ultimate compounds contain 1 to 3non-hydrogen groups R⁶.

General Method A—The key intermediates in which the rings A and B aresubstituted phenyl and phenylene respectively are conveniently preparedby use of a Friedel-Crafts reaction of a substituted biphenyl II with anactivated acyl-containing intermediate such as the succinic or glutaricanhydride derivative III or acid chloride IV in the presence of a Lewisacid catalyst such as aluminum trichloride in an aprotic solvent such as1,1,2,2-tetrachloroethane. The well known Friedel-Crafts reaction can becarried out with many alternative solvents and acid catalysts asdescribed by E. Berliner, Org. React., 5, 229 (1949) and H. Heaney,Comp. Org. Synth., 2, 733 (1991).

If the anhydride III is monosubstituted or multiplesubstituted in anunsymmetrical way, the raw product I-A often exists as a mixture ofisomers via attack of the anhydride from either of the two carbonyls.The resultant isomers can be separated into pure forms bycrystallization or chromatography using standard methods known to thoseskilled in the art.

When they are not commercially available, the succinic anhydrides IIIcan be prepared via a Stobbe Condensation of a dialkyl succinate with analdehyde or ketone (resulting in side chain R⁶), followed by catalytichydrogenation, hydrolysis of a hemiester intermediate to a diacid andthen conversion to the anhydride III by reaction with acetyl chloride oracetic anhydride. Alternatively, the hemiester intermediate is convertedby treatment with thionyl chloride or oxalyl chloride to the acidchloride IV in which R¹² is lower alkyl. For a review of the Stobbecondensation, including lists of suitable solvents and bases see W. S.Johnson and G. H. Daub, Org. React., 6, 1 (1951). This method, asapplied to the preparation of III (R⁶=H, isobutyl and H, n-pentyl), hasbeen described by D. Wolanin, et al., U.S. Pat. No. 4,771,038, Sep. 13,1988.

Method A is especially useful for the preparation of cyclic keyintermediates such as I-A-3 in which two R⁶ groups are connected in amethylene chain to form a 4-7 membered ring. Small ring (3-5 member)anhydrides are readily available only as cis isomers which yield cisinvention compounds I-A-3. The trans compounds I-A-4 are then preparedby treatment of I-A-3 with a base such as DBU in THF.

The substituted four member ring starting material anhydrides such asIII-A-1 are formed in a photochemical 2+2 reaction as shown below. Thismethod is especially useful for the preparation of compounds in whichR¹⁴ is acetoxy or acetoxy-methylene. After the subsequent Friedel-Craftsreaction the acetate can be removed by basic hydrolysis and the carboxylprotected by conversion to 2-(tri-methylsilyl)ethyl ester. The resultantintermediate with R¹⁴=CH₂OH can be converted to key intermediates withother R¹⁴ groups by using procedures described in General Method K.

The Friedel Crafts method is also useful when double bonds are foundeither between C-2 and C-3 of a succinoyl chain (from maleic anhydrideor 1-cyclopentene-1,2-dicarboxylic anhydride, for example) or when adouble bond is found in a side chain, such as in the use of itaconicanhydride as starting material to yield products in which two R⁶ groupsas found on one chain carbon together form an exo-methylene (═CH₂)group. Subsequent uses of these compounds are described in Methods D andE.

General Method B—Alternatively key intermediates can be prepared via areaction sequence involving mono-alkylation of a dialkyl malonate VIwith an alkyl halide to form intermediate VII, followed by alkylationwith a halomethyl biphenyl ketone VIII to yield intermediate IX.Compounds of structure IX are then hydrolyzed with aqueous base and thenheated to decarboxylate the malonic acid intermediate and yield I-B-2(Method B-1). By using one equivalent of aqueous base the esters I-B-2with R¹² as alkyl are obtained, and using more than two equivalents ofbase the acid compounds (R¹²=H) are obtained. Optionally, heat is notused and the diacid or acid-ester I-B-1 is obtained. Alternatively, thediester intermediate IX can be heated with a strong acid such asconcentrated hydrochloric acid in acetic acid in a sealed tube at about110° C. for about 24 hr to yield I-B-2 (R¹²=H).

Alternatively, the reaction of VI with VIII can be conducted before thatwith the alkyl halide to yield the same IX (Method B-2).

Intermediates VIII are formed from biphenyls II in a Friedel-Craftreaction with haloacetyl halides such as bromoacetyl bromide orchloroacetyl chloride. Alternatively, the biphenyl can be reacted withacetyl chloride or acetic anhydride and the resultant producthalogenated with, for example, bromine to yield intermediates VIII(X=Br).

Method B has the advantage of yielding single regio isomers whereasMethod A yields mixtures. Method B is especially useful when the sidechains R⁶ contain aromatic or heteroaromatic rings that may participatein intramolecular acylation reactions to give side products if Method Awere to be used. This method is also very useful when the R⁶ groupadjacent to the carboxyl of the final compound contains heteroatoms suchas oxygen, sulfur, or nitrogen, or more complex functions such as imiderings.

General Method C—Especially useful is the use of chiral HPLC to separatethe enantiomers of racemic key intermediate mixtures (see, for example,D. Arlt, B. Boemer, R Grosser and W. Lange, Angew. Chem. Int. Ed. Engl.30 (1991) No. 12). The key intermediates are prepared as pureenantiomers by use of a chiral auxiliary route—see, for example: D. A.Evans, Aldrichimica Acta, 15(2), 23 (1982) and other similar referencesknown to one skilled in the art.

C-1. Acid halide X is reacted with the lithium salt of chiral auxiliaryXI (R is often isopropyl or benzyl) to yield intermediate XII, which inturn is alkylated at low temperatures (typically under −50° C.) withhalo-tert-butylacetyl compound XIII to yield pure isomer XIV. The use ofopposite chirality XI yields opposite chirality XIV. Conversion of XIVto the enantiomerically pure diacid XV is accomplished by treatment withlithium hydroxide/hydrogen peroxide in THF/water, followed by acids suchas trifluoroacetic acid. The compound XV is then converted toenantiomerically pure anhydride III-A by treatment with acetyl chloride.The use of a Friedel-Crafts reaction as in method A then converts III-Ato I-C-1.

C-2. Biphenyl starling material II may also first be reacted in aFriedel-Crafts reaction as earlier described with succinic anhydridefollowed by Fisher esterification with a lower alcohol such as methanolin the presence of a strong acid such as sulfuric acid to form acylderivative I-C-2. The carbonyl group of this material is then blocked asa ketal such as that formed by treatment with1,2-bistrimethyl-silyloxyethane in the presence of a catalyst such astrimethyl-silyltriflate in a suitable solvent. Many other ketalderivatives and reaction conditions familiar to those skilled in the artcan also be used in this step. Basic hydrolysis of the ester followed byreaction of the resultant I-C-3 with XI in the presence of an amidecoupling agent such as 1-(3-dimethylaminopropyl)-3-ethylcarbodiimideyields amide I-C-4. Reaction of this chiral amide with an alkylatingagent such as alkyl or arylalkyl triflate or halide yieldsenantiomerically enriched product I-C-5 which can be converted to ketalacid I-C-6 by treatment with a weak base such as lithiumhydroxide/hydrogen peroxide and then to keto acid I-C-7 by treatmentwith an acid. These deblocking steps can be conducted in either order.

General Method D—Key intermediates in which R⁶ are alkyl- or aryl- orheteroaryl- or acyl- or heteroarylcarbonyl-thiomethylene are prepared bymethods analogous to those described in the patent publication WO90/05719. Thus substituted itaconic anhydride XVI (n=1) is reacted underFriedel-Crafts conditions to yield acid I-D-1 which can be separated bychromatography or crystallization from small amounts of isomeric I-D-5.Alternatively, I-D-5 are obtained by reaction of key intermediates I-D-4(from any of Methods A through C) with formaldehyde in the presence of abase.

Compounds I-D-1 or I-D-5 are then reacted with a mercapto derivativeXVII or XVIII in the presence of a catalyst such as potassium carbonate,ethyldiiso-butylamine, tetrabutylammonium fluoride or free radicalinitiators such as azobisisobutyronitrile (AIBN) in a solvent such asdimethylformamide or tetrahydrofurane to yield key intermediates I-D-2,I-D-3, I-D-6 or I-D-7.

General Method E—Reaction of optionally substituted maleic anhydride XIXunder Friedel-Crafts conditions with II yields key intermediate I-E-1,which in turn is reacted with either of mercapto derivatives XVII orXVIII to yield key intermediates I-E-2 or I-E-3, or with substitutedamine XX to yield key intermediate I-E-4. Esterification of I-E-1 (R⁶=H)with CH₃I/DBU followed by reagent XXI and AgF and then basic hydrolysisyields pyrrolidine key intermediate I-E-5. R¹⁴ can be various alkyl orarylalkyl groups including benzyl. Reaction of the intermediate ester(from step 2) with benzyloxycarbonyl chloride in THF at reflux followedby hydrolysis yields key intermediates in which R¹⁴ isbenzyloxycarbonyl.

General Method F—Biaryl key intermediates such as those of thisapplication may also be prepared by Suzuki or Stille cross-couplingreactions of aryl or heteroaryl metallic compounds in which the metal iszinc, tin, magnesium, lithium, boron, silicon, copper, cadmium or thelike with an aryl or heteroaryl halide or triflate(trifluoromethane-sulfonate) or the like. In the equation below eitherMet or X is the metal and the other is the halide or triflate. Pd(com)is a soluble complex of palladium such astetrakis(triphenylphosphine)-palladium(0) orbistriphenylphosphine)palladium(II) chloride. These methods are wellknown to those skilled in the art. See, for example, A. Suzuki, PureAppl. Chem., 66, 213-222 (1994); A. Suzuki, Pure Appl. Chem., 63,419-422 (1991); and V. Farina and G. Roth, “Metal-Organic Chemistry”Volume 5 (Chapter 1), 1994.

The starting materials XXIII (B=1,4-phenylene) are readily formed usingmethods analogous to those of methods A, B or C but using a halobenzenerather than a biphenyl as starting material. When desired, the materialsin which X is halo can be converted to those in which X is metal byreactions well known to those skilled in the art such as treatment of abromo intermediate with hexamethylditin and palladiumtetrakistriphenylphosphine in toluene at reflux to yield thetrimethyltin intermediate. The starting materials XXIII (B=heteroaryl)are most conveniently prepared by method C but using readily availableheteroaryl rather than biphenyl starting materials. The intermediatesXXII are either commercial or easily prepared from commercial materialsby methods well known to those skilled in the art.

These general methods are useful for the preparation of keyintermediates for which Friedel-Crafts reactions such as those ofMethods A, B, C, D or E would lead to mixtures with various biarylacylation patterns. Method F is also especially useful for thepreparation of key intermediates in which the aryl groups A or B containone or more heteroatoms (heteroaryls) such as those compounds thatcontain thiophene, furan, pyridine, pyrrole, oxazole, thiazole,pyrimidine or pyrazine rings or the like instead of phenyls.

Method F

-   -   T, x, A, B, E and D as in Structure I    -   Met=Metal and X=Halide or Triflate or    -   Met=Halide or Triflate and X=Metal

General Method G—When the R⁶ groups of method F form together a 4-7membered carbocyclic ring as in Intermediate XXV below, the double bondcan be moved out of conjugation with the ketone group by treatment withtwo equivalents of a strong base such as lithium diisopropylamide orlithium hexamethylsilylamide or the like followed by acid quench toyield compounds with the structure XXVI. Reaction of XXVI with mercaptoderivatives using methods analogous to those of General Method D thenleads to key intermediate I-G-1 or I-G-2.

General Method H—Key intermediates in which two R⁶ groups form a 4-7member carbocyclic ring as in I-H below and R¹⁴ is alkyl or arylalkylare prepared according to method H. Starting material XXVII is reactedwith two equivalents of a strong base such as lithium diisopropylamide(LDA) followed by an alkyl or arylalkyl halide (R¹⁴X) to yieldintermediate XXVIII. This material is then reduced to the alcohol with areducing agent capable of selective reduction of the ketone such assodium borohydride, followed by dehydration withtriphenylphosphine/diethyl azodicarboxylate (DEAD) in a suitable solventsuch as THF at reflux to yield XXIX. Hydrolysis of the ester withaqueous base followed by amide formation with R¹²ONHR¹² is(C₁-C₄)-alkyl, but usually CH₃) in the presence of a coupling agent suchas dicyclohexyldiimide (DCC) yields XXX. Other acyl activating groupswell known to those skilled in the art such as acid chlorides or mixedanhydrides could be used instead of XXX. Substituted biphenyl halideXXXI is reacted with an alkyl lithium such as two equivalents of t-butyllithium to yield lithiated biphenyl XXXII which is then reacted withactivated acyl compound XXX. The resultant intermediate XXXIII is thentreated with diethylaluminum cyanide to yield intermediate XXXIV whichis then hydrolyzed with aqueous acid to yield key intermediate I-H whichis purified by chromatography on silica gel to afford pure isomers.

General Method I—Key intermediates in which two R6 groups together forma pyrrolidine ring are prepared according to method I. Starting materialXXXV (L-pyroglutaminol) is reacted under acid catalysis withbenzaldehyde XXXVI (may be substituted) to yield bicyclic derivativeXXXVII. A double bond is then introduced using phenylselenenylmethodology well known to those skilled in the art to yield XXXVIII,which, in turn, is reacted with a vinylcopper (I) complex to yieldconjugate addition product XXXIX. Such reactions in which Lig can be,for example, another equivalent of vinyl group or halide are well knownto those skilled in the art. Hydride reduction (lithium aluminum hydrideor the like) of XXXIX followed by standard blocking with, for example,t-butyldimethylsilylchloride yields XXXX which in turn is reacted withan optionally substituted benzylchloroformate XXXXI to yield XXXXII.Ozonolysis of this intermediate followed by reductive workup(dimethylsulfide, zinc/acetic acid or the like) leads to aldehydeXXXXIII. Reaction of this aldehyde with a biphenyl organometallic suchas XXXII yields alcohol XXXXIV. Deblocking of the silyl group with, forexample, tetrabutylammonium fluoride followed by oxidation with, forexample, pyridiniumdichromate or the like yields key intermediate 1-I-1in which R¹⁴ is a carbobenzyloxy group.

Alternatively the carbobenzyloxy group is removed by reaction withhydrogen and a catalyst such as palladium on carbon to yield theunsubstituted key intermediate 1-I-2 optionally followed by N-alkylationto yield key intermediate 1-I-3. These final steps are well known tothose skilled in the art. Alternatively the intermediate XXXX can bedirectly treated with ozone followed by the other steps of this methodto yield 1-I-3, in which R¹⁴ is optionally substituted benzyl ratherthan as in 1-I-1.

This method is especially useful to prepare single enantiomers becausestarting material XXXV is available as either the isomer as drawn or asD-pyroglutaminol to yield enantiomeric products.

General Method J—The key intermediates in which E represents asubstituted chain of 3 carbons are prepared by method J. IntermediatesXXXXVII, if not available from commercial sources, are prepared byreaction of an activated biphenylcarboxylic acid derivative XXXXV withsubstituted acetic acid XXXXVI which has been converted to its bis-anionwith two equivalents of a strong base such as LDA followed by heating todecarboxylate the intermediate keto acid. Product XXXXVII is thentreated with methylenemalonate derivative XXXXVIII in the presence of astrong base such as sodium hydride to yield substituted malonate XXXXIX.This malonate can be further alkylated under conditions familiar tothose skilled in the art to yield L which in turn is treated with acidand then heated to yield key intermediate 1-J-1, Alternatively the finalalkylation can be omitted to yield products in which the R⁶ adjacent tothe carboxyl is H. Alternatively XXXXVII can be alkylated with3-halopropionate ester LI in the presence of base such as LDA to yieldester 1-J-2 which can then be hydrolyzed with aqueous base to yield keyintermediate 1-J-3 upon treatment with acid. This method is especiallyuseful if any of the groups R⁶ contain aromatic residues.

Method K—The key intermediates in which two R⁶ groups are joined to forma substituted 5-member ring are most conveniently prepared by method K.In this method acid LII (R=H) is prepared using the protocols describedin Tetrahedron, Vol. 37, Suppl., 1981, 411. The acid is protected as anester (R=benzyl or 2-(trimethylsilyl)ethyl) by use of coupling agentssuch as 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride andprocedures well known to those skilled in the art. Substitutedbromobiphenyl LIII is converted to its Grignard reagent by treatmentwith magnesium which is then reacted with LII to yield alcohol LIV.Alcohol LIV is eliminated via base treatment of its mesylate by usingconditions well known to those skilled in the art to yield olefin LV.Alternatively LIII is converted to a trimethyltin intermediate viainitial metallation of the bromide with n-butyllithium at lowtemperature (−78° C.) followed by treatment with chlorotrimethyltin andLII is converted to an enoltriflate by reaction with2-[N,N-bis(trifluoromethylsulfonyl)-amino]-5-chloropyridine in thepresence of a strong aprotic base. The tin and enoltriflateintermediates are then coupled in the presence of a Pd^(o) catalyst, CuIand AsPh₃ to yield directly intermediate LV. Ozonolysis of LV (workupwith methyl sulfide) yields aldehyde LVI. Alternatively treatment withOsO₄ followed by HIO₄ converts LV to LVI.

Conversion of intermediate LVI to key intermediate I-K is accomplishedin several ways depending on the identity of side chain function X.Reaction of LVI with Wittig reagents followed by hydrogenation yieldsproducts in which X is alkyl, aryl or arylalkyl. Reduction of aldehydeLVI with LAH yields alcohol I-K (X=OH). The alcohol is converted tophenyl ethers or N-phthalimidoyl compounds by use of the appropriatestarting materials and Mitsunobu conditions well known to those skilledin the art; see O. Mitsunobu, Synthesis, 1 (1981). Alternatively thealcohol of I-K (X=OH) is converted to a leaving group such as tosylate(X=OTs) or bromide (X=Br) by conditions well known to those skilled inthe art and then the leaving group is displaced by sulfur or azidenucleophiles to yield products with X=thioether or azide which in turnis reduced and acylated to yield amides (X=NHAcyl). Direct acylation ofthe alcohol I-K (X=OH) yields key intermediates in which X=OAcyl andreaction of the alcohol with various alkyl halides in the presence ofbase yields alkyl ethers (X=OR²). In each case a final step is removalof acid blocking group R to yield acids (R=H) by using conditions whichdepend on the stability of R and X, but in all cases well known to thoseskilled in the art such as removal of benzyl by base hydrolysis or of2-(trimethylsilyl)ethyl by treatment with tetrabutylammonium fluoride.

Suitable pharmaceutically acceptable salts of the compounds of thepresent invention that contain an acidic moiety include addition saltsformed with organic or inorganic bases. The salt forming ion derivedfrom such bases can be metal ions, e.g., aluminum, alkali metal ions,such as sodium of potassium, alkaline earth metal ions such as calciumor magnesium, or an amine salt ion, of which a number are known for thispurpose. Examples include ammonium salts, arylalkylamines such asdibenzylamine and N,N-dibenzylethylenediamine, lower alkylamines such asmethylamine, t-butylamine, procaine, lower alkylpiperidines such asN-ethyl-piperidine, cycloalkylamines such as cyclohexylamine ordicyclohexylamine, 1-adamantylamine, benzathine, or salts derived fromamino acids like arginine, lysine or the like. The physiologicallyacceptable salts such as the sodium or potassium salts and the aminoacid salts can be used medicinally as described below and are preferred.

Suitable pharmaceutically acceptable salts of the compounds of thepresent invention that contain a basic moiety include addition saltsformed with organic or inorganic acids. The salt forming ion derivedfrom such acids can be halide ions or ions of natural or unnaturalcarboxylic or sulfonic acids, of which a number are known for thispurpose. Examples include chlorides, acetates, tartrates, or saltsderived from amino acids like glycine or the like. The physiologicallyacceptable salts such as the chloride salts and the amino acid salts canbe used medicinally as described below and are preferred.

These and other salts which are not necessarily physiologicallyacceptable are useful in isolating or purifying a product acceptable forthe purposes described below.

The salts are produced by reacting the acid form of the inventioncompound with an equivalent of the base supplying the desired basic ionor the basic form of the invention compound with an equivalent of theacid supplying the desired acid ion in a medium in which the saltprecipitates or in aqueous medium and then lyophilizing. The free acidor basic form of the invention compounds can be obtained from the saltby conventional neutralization techniques, e.g., with potassiumbisulfate, hydrochloric acid, sodium hydroxide, sodium bicarbonate, etc.

The compounds of the present invention are expected to inhibit thematrix metalloproteases MMP-2, MMP-3, MMP-8, MMP-9, MMP-12, MMP-13, andthe related protease TACE, as well as the release of TNFα in vivo, andare therefore expected to be useful for treating or preventing theconditions referred to in the background section. As other MMPs notlisted above share a high degree of homology with those listed above,especially in the catalytic site, it is deemed that compounds of theinvention should also inhibit such other MMPs to varying degrees.

Varying the substituents on the biaryl portions of the molecules, aswell as those of the R⁶ groups of the claimed compounds, is expected toaffect the relative inhibition of the listed MMPs. Thus compounds ofthis general class can be “tuned” by selecting specific substituentssuch that inhibition of specific MMP(s) associated with specificpathological conditions can be enhanced while leaving non-involved MMPsless affected.

The compounds of the present invention exhibit good activity for MMP-2,MMP-3, MMP-8, MMP-9, MMP-12 and MMP-13, and a good selectivity for theseMMP's over other MMP's such as MMP-1 and MMP-7.

As a result of the abovementioned selectivity profile, the compounds ofthe present invention are especially suitable for the treatment ofrespiratory diseases.

The method of treating matrix metalloprotease-mediated or TNFαrelease-mediated conditions may be practiced in mammals, includinghumans, which exhibit such conditions.

The inhibitors of the present invention are contemplated for use inveterinary and human applications. For such purposes, they will beemployed in pharmaceutical compositions containing active ingredient(s)plus one or more pharmaceutically acceptable carriers, diluents,fillers, binders, and other excipients, depending on the administrationmode and dosage form contemplated.

Administration of the inhibitors may be by any suitable mode known tothose skilled in the art. Examples of suitable parenteral administrationinclude intravenous, intraarticular, subcutaneous and intramuscularroutes. Intravenous administration can be used to obtain acuteregulation of peak plasma concentrations of the drug. Improved half-lifeand targeting of the drug to the joint cavities may be aided byentrapment of the drug in liposomes. It may be possible to improve theselectivity of liposomal targeting to the joint cavities byincorporation of ligands into the outside of the liposomes that bind tosynovial-specific macromolecules. Alternatively intramuscular,intraarticular or subcutaneous depot injection with or withoutencapsulation of the drug into degradable microspheres e.g., comprisingpoly(DL-lactide-co-glycolide) may be used to obtain prolonged sustaineddrug release. For improved convenience of the dosage form it may bepossible to use an i.p. implanted reservoir and septum such as thePercuseal system available from Pharmacia Improved convenience andpatient compliance may also be achieved by the use of either injectorpens (e.g. the Novo Pin or Q-pen) or needle-free jet injectors (e.g.from Bioject, Mediject or Becton Dickinson). Prolonged zero-order orother precisely controlled release such as pulsatile release can also beachieved as needed using implantable pumps with delivery of the drugthrough a cannula into the synovial spaces. Examples include thesubcutaneously implanted osmotic pumps available from ALZA, such as theALZET osmotic pump.

Nasal delivery may be achieved by incorporation of the drug intobioadhesive particulate carriers (<200 μm) such as those comprisingcellulose, polyacrylate or polycarbophil, in conjunction with suitableabsorption enhancers such as phospholipids or acylcarnitines. Availablesystems include those developed by DanBiosys and Scios Nova.

Oral delivery may be achieved by incorporation of the drug into tablets,coated tablets, dragées, hard and soft gelatine capsules, solutions,emulsions or suspensions. Oral delivery may also be achieved byincorporation of the drug into enteric coated capsules designed torelease the drug into the colon where digestive protease activity islow. Examples include the OROS-CT/Osmet™ and PULSINCAP™ systems fromALZA and Scherer Drug Delivery Systems respectively. Other systems useazocrosslinked polymers that are degraded by colon specific bacterialazoreductasas, or pH sensitive polyacrylate polymers that are activatedby the rise in pH at the colon. The above systems may be used inconjunction with a wide range of available absorption enhancers.

Rectal delivery may be achieved by incorporation of the drug intosuppositories.

The compounds of this invention can be manufactured into the abovelisted formulations by the addition of various therapeutically inert,inorganic or organic carriers well known to those skilled in the art.Examples of these include, but are not limited to, lactose, corn starchor derivatives thereof, talc, vegetable oils, waxes, fats, polyols suchas polyethylene glycol, water, saccharose, alcohols, glycerin and thelike. Various preservatives, emulsifiers, dispersants, flavorants,wetting agents, antioxidants, sweeteners, colorants, stabilizers, salts,buffers and the like are also added, as required to assist in thestabilization of the formulation or to assist in increasingbioavailability of the active ingredient(s) or to yield a formulation ofacceptable flavor or odor in the case of oral dosing.

The amount of the pharmaceutical composition to be employed will dependon the recipient and the condition being treated. The requisite amountmay be determined without undue experimentation by protocols known tothose skilled in the art Alternatively, the requisite amount may becalculated, based on a determination of the amount of target enzymewhich must be inhibited in order to treat the condition. It is expectedthat the compounds of the invention generally will be administered indoses in the range of 0.01-100 mg per kg of body weight per day.

The matrix metalloprotease inhibitors of the invention are useful notonly for treatment of the physiological conditions discussed above, butare also useful in such activities as purification of metalloproteasesand testing for matrix metalloprotease activity. Such activity testingcan be both in vitro using natural or synthetic enzyme preparations orin vivo using, for example, animal models in which abnormal destructiveenzyme levels are found spontaneously (use of genetically mutated ortransgenic animals) or are induced by administration of exogenous agentsor by surgery which disrupts joint stability.

Biological Protocols

Inhibitory activities of the compounds of the invention against matrixmetallo-proteases and production of TNFα may be determined as describedbelow.

P218 Quenched Fluorescence Assay for MMP Inhibition:

This assay is adapted from the one described by Knight et al., FEBSLetters, 296, 263-266 (1992) for MMP-3 and a related substrate. The rateof hydrolysis of the synthetic substrateH-MCA-Pro-Lys-Pro-Leu-Ala-Leu-DPA-Ala-Arg-NH₂ (P218) by the respectiveMMPs is monitored fluorometrically, using an excitation wavelength of340 nm and an emission wavelength of 395 nm, in the presence or absenceof the test compounds. The substrate is made up initially in 100% DMSOto a concentration of 1×10⁻² M, then diluted in assay buffer to a finalconcentration of 20 μM. Test compounds (10 mM in DMSO) are diluted inassay buffer at an initial concentration of 0.3-1000 nM. These arediluted to a final concentration in the assay from 0.03 nM to 100 nM.The reaction is initiated by the addition of substrate at a finalconcentration of 20 μM. The total assay volume in a 96 well microtitreplate is 150 μl, Cleavage of the substrate between the Leu-Ala residuesallows the fluorescence of the MCA group to be detected on a fluorometer(Cytofluor II) following excitation at 340 nm and emission at 395 nm.Change in fluorescence is continually monitored for a 40 min period.

The K_(i)'s are calculated using the method described by Williams andMorrison, Methods in Enzymology, 63, 437-467 (1979) to measureK_(i apparent) for tight binding inhibitors, and is summarised asfollows:[I] ₀/(1−v _(i) /v ₀)=K _(i apparent) ×v _(i) /v ₀ +[E] ₀[I]₀ and [E]₀ are inhibitor and enzyme concentrations, and v_(i)/v₀ arereaction velocities with/without inhibitor. [I]₀ is equal to IC₅₀ whenv_(i) is half v₀, so that:IC₅₀=0.5×[E] ₀ +K _(i apparent)

IC₅₀'s are determined at each enzyme concentration using Xlfit software.K_(i apparent) is then determined graphically from the plot of IC₅₀versus MMP concentration, using the intercepts to estimateK_(i apparent). Thus, intercept values at IC₅₀=0 and [E]₀=0 are equal to−2×K_(i apparent) and K_(i apparent), respectively. IC₅₀ values arecalculated using % inhibition values at each enzyme concentration,ensuring data is taken from the linear part of the reaction rate curves.The K_(i) can then be calculated from the equation:K _(i) =K _(i apparent)/(1+S)/K _(m)where S=substrate concentration and K_(m)=dissociation constant.

The assay conditions are modified as follows for each of the MMPs used:

MMP-1 (Human Gingival Fibroblast Interstitial Collagenase)

Pro-MMP-1 was supplied by Jack Windsor [Windsor et al., J. Biol. Chem.269 (42), 26201-26207 (1994)]. The pro enzyme was activated byincubating in 1:20 Trypsin/1 mM AEBSF for 10 min at 25° C.

-   -   K_(m) value: 30 μM    -   Assay Buffer: The assay is carried out in buffer containing 50        mM HEPES, 10 mM CaCl₂, pH 7.0.    -   Enzyme concentrations for IC₅₀ determinations: 0.1 μg/ml    -   Enzyme concentrations for K_(i) determinations: 0.1-0.8 μg/ml

MMP-2 (Gelatinase A)

Gelatinase A (MMP-2) is prepared using a vaccinia expression systemaccording to the method of R. Fridman et al., J. Biol. Chem., 267, 15398(1992).

-   -   Assay Buffer; The assay is carried out in buffer containing 50        mM Tris, 150 mM NaCl, 10 mM CaC₂, 0.005% Brij-35 at pH 7.0.    -   Enzyme concentrations for IC₅₀ determinations: 0.078 μg/ml

MMP-3 (Stromelysin)

Preparation of Recombinant Truncated Pro-stromelysin (MMP-3):

Truncated Pro-stromelysin-257 is expressed in a soluble form in E. colias described by Marcy et al., Biochemistry, 30, 6476-6483, 1991 (seealso Cancer Treat. Res. 61, 21-41 (1992)). Soluble truncatedprostromelysin is purified by a modification of the monoclonal antibodyaffinity chromatography method described by Housley et al., J. Biol.Chem., 268, 4481-87, 1993.

-   -   Assay Buffer: The assay is carried out in buffer containing 50        mM HEPES, 10 mM CaCl₂, pH 7.0.    -   Enzyme concentrations for IC₅₀ determinations; 0.8 μg/ml

MMP-7 (Matrilysin)

Catalytic domain expressed in E. coli using the pET 14 vector, providedby Dr Steve Shapiro, Jewish Hospital at the Washington UniversityMedical Center, St. Louis, Mo., USA.

-   -   Assay Buffer: The assay is carried out in buffer containing 50        mM HEPES, 10 mM CaCl₂, pH 7.0.    -   Enzyme concentrations for IC₅₀ determinations: 0.3 μg/ml

MMP-8 (Neutrophil Collagenase)

Active recombinant truncated form (met⁸⁰-gly²⁴²) was obtained accordingto the protocol of Knauper, Eur. J. Biochem. 189, 296-300 (1990).

-   -   Assay Buffer: The assay is carried out in buffer containing 50        mM HEPES, 10 mM CaCl₂, pH 7.0.    -   Enzyme concentrations for IC₅₀ determinations: 9.4 μg/ml.

MMP-9 (Gelatinase B)

MMP-9 is isolated modifying the previously described procedures of Hibbset al. (J. Biol. Chem., 260, 2493-2500, 1984) and Wilhelm et al. (J.Biol. Chem., 264, 17213-17221, 1989). Briefly, polymorphonuclearleukocytes (PMN) preparations are isolated as described above from 3 ormore units of freshly drawn whole blood. Cells are resuspended inphosphate buffered saline (PBS) containing 100 ng/ml phorbol myristateacetate (PMA) in the presence of 50 mM di-isopropylfluorophospate (DFP),1 μg/ml leupeptin and aprotinin, and 1 mg/ml catalase for 1 hr at 37° C.Supernatants are collected by centrifugation (300×g) and the samples arefrozen at −70° C. All chromatographic methods are performed at 4° C.Thawed samples are concentrated 5-fold using an Amicon chamber equippedwith a YM-10 membrane. The concentrate is pressure dialyzed against0.02M Tris-HCl, 0.1 M NaCl, 1 mM CaCl₂, 1 μM ZnCl₂, 0.001% Brij-35,0.02% sodium azide (NaN₃), pH 7.5 and applied to DEAE ion exchangechromatography resin which is previously equilibrated with the samebuffer at a flow rate of 0.4 ml/min. The column is extensively washedwith the same buffer and gelatinase is eluted as 4 ml fractions from thecolumn with 0.02M Tris-HCl, 0.5 M NaCl, 1 mM CaCl₂, 1 μM ZnCl₂, 0.001%Brij-35, 0.02% NaN₃, pH 7.5. Gelatinase containing fractions areobserved by gelatin zymography (see below), loaded onto a gelatinagarose affinity resin and washed with the same buffer. Gelatinaseactivity is eluted at a flow rate of 1 ml/min from the column as 1 mlfractions with 0.02M Tris-HCl, 1 M NaCl, 1 mM CaCl₂, 1 μM ZnCl₂, 0.001%Brij-35, 0.02% NaN₃, pH 7.5 containing 10% dimethyl sulfoxide (DMSO).The fractions containing gelatinase activity are pooled and dialyzedagainst 0.005M Tris-HCl, 5 mM NaCl, 0.5 mM CaCl₂, 0.1 μMZnCl_(2, 0.001)% Brij-35, pH 7.4. The protein content associated withmaterial is determined with a micro-BCA assay (Pierce, Rockford, Ill.),lyophilized and reconstituted to a desired working concentration (100μg/ml). Recombinant human pro-MMP-9 is also expressed in baculovirus.

-   -   K_(m) value: 22 μM    -   Assay Buffer: The assay is carried out in buffer containing 50        mM HEPES, 150 mM NaCl, 10 mM CaCl, 0.005% Brij-35, pH 7.0.    -   Enzyme concentrations for IC₅₀ determinations: 0.38 μg/ml    -   Enzyme concentrations for K_(i) determinations: 0.38-1.00 μg/ml

MMP-12 (Macrophage Elastase)

MMP-9 is isolated according to the following procedure: Human GenePoolcDNA libraries from human lung, spleen, and brain were obtained fromInvitrogen (Groningen, The Netherlands). HPLC-purified oligonucleotideswere purchased from BioTez (Berlin, Germany), E. coli strain DH5α fromLife Technologies (Heidelberg, Germany), strain BL21(DE3) from Novagen(Heidelberg, Germany). Pfu DNA-Polymerase and pBlueScriptII-KS(+) werefrom Stratagene (Heidelberg, Germany). DNA plasmid and DANN gelisolation kits were purchased from Qiagen (Hilden, Germany). Allrestriction enzymes and DNA modifying enzymes were ordered from NewEngland Biolabs (Schwalbach, Germany). HiTrapQ (5 ml) was from PharmaciaBiotech (Freiburg, Germany). Vydac C4-RP HPLC columns (214TP54: 300 Å, 5μm, 4.6×250 mm; 214TP1022: 300 Å, 10 μm, 22×250 mm) were from Promochem(Wesel, Germany). Fluorogenic substrate P218 was purchased fromPolypeptide Laboratories (Wolfenbüttel, Germany). Ultrapure urea waspurchased from Schwarz-Mann (Cleveland, Ohio). Ready-to-use NuPAGE 10%Bis-Tris Polyacrylamide SDS-PAGE gels and NuPAGE 20×MOPS running bufferwere obtained from Novex (Frankfurt/Main, Germany) 10% AMPA and allother chemicals were from Sigma (Deisenhofen, Germany).

Molecular Cloning of MMP-12: The CDS for human MMP-12 (GenBank accessionnumber: L23808; SwissProt accession number: P39900) was cloned by PCRout of the human normal spleen cDNA library with the gene-specificprimers Oligo-428 (forward primer with BamH1-site underlined: 5′-aaa tttaaa gga tcc gcc acc atg aag ttt ctt cta ata ctg ctc ctg-3′) andOligo-431 (reverse primer with EcoR1-site underlined: 5′-aaa ttt aaa gaattc att aac aac caa acc agc tat tgc ttt tca-3′). Removal of an internalEcoR1-site was done by PCR-mutagenesis with Oligo-434 (mutated baseunderlined: 5′-gcc tcc tga atg tgt agt cca gaa ctc gtc ctc atc gaa atgtgc atc-3′. The PCR reaction (100 μl volume) was carried out with PfuDNA-polymerase in a Perkin Elmer GeneAmp 2400 PCR system with 25 cyclesof denaturation (94° C., 1 min), annealing (60° C., 1 min) and extension(72° C., 2 min). The PCR product was gel purified by electrophoresis on0.8% agarose/EthBr gels, separated from the agarose gel with the QiaEx2DNA-isolation kit, cleaved with the restriction endonucleasesBamH1/EcoR1, and purified again. This material was ligated into thecloning vector pBlueScriptII-KS(+), which was previously digested withBamH1 and EcoR1-cut and dephosphorylated with calf intestine alkalinephosphatase. The recombinant plasmid (PMYZ180) was transformed into E.coli strain DH5α. Plasmid DNA from several clones was isolated with theQiagen plasmid preparation kit and analyzed by restriction digest. TheDNA sequence of the MMP-12 complete CDS was determined by the dideoxysequencing with dye terminator chemistry on an ABI 377 sequencer.Sequence analysis and all subsequent bioinformatic work was done withthe software package Lasergene from DNAStar (Madison, Wis.). Sequenceanalysis of construct pMYZ 180 (4369 bp) identified silent mutations inthe CDS of MMP-12 at positions 802 (G→A), 1402 (T→C), 1429 (C→T) and2002 (T→C), which left the amino acid sequence of the proteinunaffected.

Construction of Expression Vectors: In order to test the best strategyfor both protein expression and protein purification, several differentexpression constructs with either full-length or partial sequences ofthe MMP-12 gene plus various affinity tags for purification weregenerated by PCR, The following constructs were made:

pMYZ187: The coding sequence for full-length human MMP-12 was cleavedvia BamH1/EcoR1 digest out of pMYZ180, ligated into BamH1/EcoR1 cleavedpET-28a, and the complete coding sequence of final construct wasverified by double-stranded DNA-sequencing.

pMYZ188: The coding sequence for full-length human MMP-12 was cleavedvia BamH1/EcoR1 digest out of pMYZ180, ligated into BamH1/EcoR1 cleavedpET-32a, and the complete coding sequence of final construct wasverified by double-stranded DNA-sequencing.

pMYZ189: The coding sequence for full-length human MMP-12 was cleavedvia BamH1/EcoR1 digest out of pMYZ180, ligated into BamH1/EcoR1 cleavedpMYZ173 (a derivative of pET28a with an aminoterminal tag of the GB1domain), and the complete coding sequence of final construct wasverified by double-stranded DNA-sequencing.

pMYZ194: The coding sequence for amino acids 1-279 of human MMP-12 wasamplified via PCR with primers Oligo-428 (forward primer with BamH1-siteunderlined: 5′-aaa ttt aaa gga tcc gcc acc atg aag ttt ctt cta ata ctgctc ctg-3′) and Oligo-486 (reverse primer with EcoR1-site underlined:5′-ttt aaa ttt gaa ttc att atg gtt ctg aat tgt cag gat ttg gca-3′) outof pMYZ180, cleaved with BamH1/EcoR1, ligated into BamH1/EcoR1 cleavedpET-28a, and the complete coding sequence of final construct wasverified by double-stranded DNA-sequencing.

pMYZ195: The coding sequence for amino acids 1-264 of human MMP-12 wasamplified via PCR with primers Oligo-428 (forward primer with BamH1-siteunderlined: 5′-aaa ttt aaa gga tcc gcc acc atg aag ttt ctt cta ata ctgctc ctg-3′) and Oligo-487 (reverse primer with EcoR1-site underlined:5′-ttt aaa ttt gaa ttc att agt ctc cat aca ggg act gaa tgc cac-3′) outof pMYZ180, cleaved with BamH1/EcoR1, ligated into BamH1/EcoR1 cleavedpET-28a, and the complete coding sequence of final construct wasverified by double-stranded DNA-sequencing.

pMYZ198: The coding sequence for amino acids 1-279 of human MMP-12 wasamplified via PCR with primers Oligo-428 (forward primer with BamH1-siteunderlined: 5′-aaa ttt aaa gga tcc gcc acc atg aag ttt ctt cta ata ctgctc ctg-3′) and Oligo-486 (reverse primer with EcoR1-site underlined:5′-ttt aaa ttt gaa ttc att atg gtt ctg aat tgt cag gat ttg gca-3′) outof pMYZ180, cleaved with BamH1/EcoR1, ligated into BamH1/EcoR1 cleavedpET-32a, and the complete coding sequence of final construct wasverified by double-stranded DNA-sequencing.

pMYZ199: The coding sequence for amino acids 1-264 of human MMP-12 wasamplified via PCR with primers Oligo-428 (forward primer with BamH1-siteunderlined: 5′-aaa ttt aaa gga tcc gcc acc atg aag ttt ctt cta ata ctgctc ctg-3′) and Oligo-487 (reverse primer with EcoR1-site underlined:5′-ttt aaa ttt gaa ttc att agt ctc cat aca ggg act gaa tgc cac-3′) outof pMYZ180, cleaved with BamH1/EcoR1, ligated into BamH1/EcoR1 cleavedpET-32a, and the complete coding sequence of final construct wasverified by double-stranded DNA-sequencing.

pMYZ200: The coding sequence for amino acids 1-279 of human MMP-12 wasamplified via PCR with primers Oligo-428 (forward primer with BamH1-siteunderlined: 5′-aaa ttt aaa gga tcc gcc acc atg aag ttt ctt cta ata ctgctc ctg-3′) and Oligo-486 (reverse primer with EcoR1-site underlined:5′-ttt aaa ttt gaa ttc att atg gtt ctg aat tgt cag gat ttg gca-3′) outof pMYZ180, cleaved with BamH1/EcoR1, ligated into BamH1/EcoR1 cleavedpMYZ173, and the complete coding sequence of final construct wasverified by double-stranded DNA-sequencing.

pMYZ201: The coding sequence for amino acids 1-264 of human MMP-12 wasamplified via PCR with primers Oligo-428 (forward primer with BamH1-siteunderlined: 5′-aaa ttt aaa gga tcc gcc acc atg aag ttt ctt cta ata ctgctc ctg-3′) and Oligo-487 (reverse primer with EcoR1-site underlined:5′-ttt aaa ttt gaa ttc att agt ctc cat aca ggg act gaa tgc cac-3′) outof pMYZ180, cleaved with BamH1/EcoR1, ligated into BamH1/EcoR1 cleavedpMYZ173, and the complete coding sequence of final construct wasverified by double-stranded DNA-sequencing.

pMYZ215: The coding sequence for amino acids 17-279 of human MMP-12 wasamplified via PCR with primers Oligo-513 (forward primer with Nde1-siteunderlined: 5′-aaa ttt aaa cat atg ctt ccc ctg aac agc tct aca agcctg-3′) and Oligo-486 (reverse primer with EcoR1-site underlined: 5′-tttaaa ttt gaa ttc att atg gtt ctg aat tgt cag gat ttg gca-3′) out ofpMYZ180, cleaved with Nde1/EcoR1, ligated into Nde1/EcoR1 cleavedpET-28a, and the complete coding sequence of final construct wasverified by double-stranded DNA-sequencing.

pMYZ216: The coding sequence for amino acids 17-264 of human MMP-12 wasamplified via PCR with primers Oligo-513 (forward primer with Nde1-siteunderlined: 5′-aaa ttt aaa cat atg ctt ccc ctg aac agc tct aca agcctg-3′) and Oligo-487 (reverse primer with EcoR1-site underlined: 5′-tttaaa ttt gaa ttc att agt ctc cat aca ggg act gaa tgc cac-3′) out ofpMYZ180, cleaved with Nde1/EcoR1, ligated into Nde1/EcoR1 cleavedpET-28a, and the complete coding sequence of final construct wasverified by double-stranded DNA-sequencing.

pMYZ217: The coding sequence for amino acids 17-279 of human MMP-12 wasamplified via PCR with primers Oligo-514 (forward primer with Nco1-siteunderlined: 5′-aaa ttt aaa gcc atg gct ctt ccc ctg aac agc tct aca agcctg-3′) and Oligo-486 (reverse primer with EcoR1-site underlined: 5′-tttaaa ttt gaa ttc att atg gtt ctg aat tgt cag gat ttg gca-3′) out ofpMYZ180, cleaved with Nco1/EcoR1, ligated into Nco1/EcoR1 cleavedpET-32a, and the complete coding sequence of final construct wasverified by double-stranded DNA-sequencing.

pMYZ218: The coding sequence for amino acids 17-264 of human MMP-12 wasamplified via PCR with primers Oligo-514 (forward primer with Nco1-siteunderlined: 5′-aaa ttt aaa gcc atg gct ctt ccc ctg aac agc tct aca agectg-3′) and Oligo-487 (reverse primer with EcoR1-site underlined: 5′-tttaaa ttt gaa ttc att agt ctc cat aca ggg act gaa tgc cac-3′) out ofpMYZ180, cleaved with Nco1/EcoR1, ligated into Nco1/EcoR1 cleavedpET-32a, and the complete coding sequence of final construct wasverified by double-stranded DNA-sequencing.

pMYZ219: The coding sequence for amino acids 17-279 of human MMP-12 wasamplified via PCR with primers Oligo-515 (forward primer with BamH1-siteunderlined: 5′-aaa ttt aaa gga tcc ctt ccc ctg aac age tct aca agectg-3′) and Oligo-486 (reverse primer with EcoR1-site underlined: 5′-tttaaa ttt gaa ttc att atg gtt ctg aat tgt cag gat ttg gca-3′) out ofpMYZ180, cleaved with BamH1/EcoR1, ligated into BamH1/EcoR1 cleavedpMYZ173, and the complete coding sequence of final construct wasverified by double-stranded DNA-sequencing.

pMYZ220: The coding sequence for amino acids 17-264 of human MMP-12 wasamplified via PCR with primers Oligo-515 (forward primer with BamH1-siteunderlined: 5′-aaa ttt aaa gga tcc ctt ccc ctg aac agc tct aca agcctg-3′) and Oligo-487 (reverse primer with EcoR1-site underlined: 5′-tttaaa ttt gaa ttc att agt ctc cat aca ggg act gaa tgc cac-3′) out ofpMYZ180, cleaved with BamH1/EcoR1, ligated into BamH1/EcoR1 cleavedpMYZ173, and the complete coding sequence of final construct wasverified by double-stranded DNA-sequencing.

pMYZ227: The coding sequence for amino acids 100-279 of human MMP-12 wasamplified via PCR with primers Oligo-533 (forward primer with Nde1-siteunderlined: 5′-aaa ttt aaa cat atg ttc agg gaa atg cca ggg ggg ccc gtatgg-3′) and Oligo-486 (reverse primer with EcoR1-site underlined: 5′-tttaaa ttt gaa ttc att atg gtt ctg aat tgt cag gat ttg gca-3′) out ofpMYZ180, cleaved with Nde1/EcoR1, ligated into Nde1/EcoR1 cleavedpET-29a, and the complete coding sequence of final construct wasverified by double-stranded DNA-sequencing.

pMYZ228: The coding sequence for amino acids 100-264 of human MMP-12 wasamplified via PCR with primers Oligo-533 (forward primer with Nde1-siteunderlined: 5′-aaa ttt aaa cat atg ttc agg gaa atg cca ggg ggg ccc gtatgg-3′) and Oligo-487 (reverse primer with EcoR1-site underlined: 5′-tttaaa ttt gaa ttc att agt ctc cat aca ggg act gaa tgc cac-3′) out ofpMYZ180, cleaved with Nde1/EcoR1, ligated into Nde1/EcoR1 cleavedpET-28a, and the complete coding sequence of final construct wasverified by double-stranded DNA-sequencing.

Bacterial Expression: Batches of one liter of LB medium (10 g tryptone,5 g yeast extract, 10 g NaCl) containing ampicillin (200 μg/ml) orkanamycin (100 μg/ml) were inoculated with 20 ml each of overnightculture of E. coli BL21 (DE3) cells with the appropriate expressionvector. Cells were cultured at 37° C. to an OD600 of about 0.8 beforeinduction with IPTG (1 mM final concentration). Incubation was continuedat 37° C. for 4 hours before harvesting by centrifugation. Cell pelletswere frozen immediately and kept at −20° C. until use. Aliquots (100 μl)were taken from each cell culture and analyzed for protein expression on10% SDS-PAGE gels.

Protein Purification: Frozen cell pellets from 1 liter bacterial cellswere thawed, dissolved in 50 ml Tris/HCl, pH 8.0 with 15% glycerol,sonicated with 4 pulses a 10 sec and centrifuged for 30 min at 20,000rpm with a JA-20 rotor. After removal of the supernatant, the inclusionbodies in the pellet were dissolved over night at room temperature with50 ml 8M urea, 50 mM Tris/HCl, pH 8.0 upon gentle shaking. The next day,the solution was centrifuged for 30 min at 20,000 rpm with a JA-20 rotorand the supernatant used for further purification. Batches of 5-10 mlwere applied to a 5 ml HiTrapQ ion exchange column running in 8M urea,50 mM Tris/HCl, pH 8.0 (buffer A). After sample loading and washing ofthe column, protein was eluted by increasing the salt concentration witha solution of 8M urea, 50 mM Tris/HCl, pH 8.0+1.0 M NaCl (buffer B) in alinear gradient of 0-50% buffer B within 40 column volumes. Fractionswith protein elution at around 30% buffer B were tested for purity bydenaturing SDS-PAGE and mass spectrometry, and the fractions containingMMP-12 were pooled together. From this solution, 5-10 ml batches weretaken, acidified to a final concentration of 10% acetic acid, andinjected onto a preparative C4-RP-HPLC column (Vydac 214TP1022: 300 Å,10 μm, 22×250 mm) with a flow rate of 10 ml/min on a Waters HPLCchromatography workstation. Starting buffer was H₂O/0.1% TFA (buffer A),and elution buffer was 90% CH₃CN/10% H₂O/0.1% TEA (buffer B). The pureprotein eluted as a single peak at around 35% buffer B and the proteincontaining fractions were again analyzed by denaturing SDS-PAGE andmass-spectrometry. Pooled fractions with MMP-12 protein were frozen inliquid N₂ and freeze-dried for 3-5 days. Lyophilized protein was storedat −20° C.

-   -   K_(m) value: 5.4 μM    -   Assay Buffer: The assay is carried out in buffer containing 50        mM HEPES and 10 mM CaCl₂, pH 7.0.    -   Enzyme concentrations for IC₅₀ determinations: 0.3 μg/ml    -   Enzyme concentrations for K_(i) determinations: 0.044-0.98 μg/ml

MMP-13 (Human Collagenase 3)

Rat pro-MMP-13 was obtained according to the protocol of Roswit, Arch.Biochem. Biophys. 225, 285-295 (1983) and activated by incubating in1:10 Trypsin.

-   -   Assay Buffer: The assay is carried out in buffer containing 20        mM Tris pH 7.5, 250 mM NaCl, 5 mM CaCl₂, 0.05% NaN₃, 0.005% Brij    -   Enzyme concentrations for IC₅₀ determinations: 0.3 μg/ml

IC₅₀-values of selected compounds are given in the following table 3.The compound numbers refer to the compounds as depicted in table 1:

Compound No. IC₅₀ MMP-12 [nM] C-III 2.8 C-V 1.7 C-XVIII 1.0 C-XXII 1.6C-XXX 4.5

The following data illustrate the selectivity of examples of theinvention for MMP-2, MMP-3, MMP-8, MMP-9, MMP-12 and MMP-13. The examplenumbers refer to the examples as described in the experimental part:

TABLE 4 Potency [K_(i), nM] vs. MMP-x Example Species 1 2 3 7 8 9 12 134 rat 296* human  639 0.5  3.7 >3000 7.5 0.7 0.3  5.6 2 rat  69*human >3000 3.2* 60* 38% inh. 21.6* 1.2 0.03  70* at 30 nM *IC₅₀ [nM]In vitro Functional Tests

-   -   1. Human alveolar macrophages    -    Human alveolar macrophages were obtained by bronchoscopy of        healthy smoking volunteers. Cells were spun and resuspended at        2×10⁶/ml. Trafficking of alveolar macrophages (+or        −lipopolysaccharide 2.5 μg.ml) across an artificial basement        membrane (Matrigel) was induced by human MCP-1 (5 ng/ml) over a        48-98 h period.        (1α,2β,5β)-2-{[4′-chloro(1,1′-biphenyl)-4-yl]carbonyl}-5-[(1,3-dihydro-1,3-dioxo-2H-isoindol-2-yl)methyl]-cyclopentane        carboxylic acid inhibited the MCP-1-induced trafficking of human        alveolar macrophages across this artificial basement membrane        (IC₅₀≦1 μM).    -   2. Murine peritoneal macrophages    -    Murine macrophages were obtained from mice 5 days after an        intraperitoneal injection of thioglycollate. Cells were spun and        resuspended at 2×10⁶/ml. Trafficking of peritoneal macrophages        across an artificial basement membrane (Matrigel) was induced by        murine MCP-1 (5 ng/ml) over a 48-98 h period.        (1α,2β,5β)-2-{[4′-chloro(1,1′-biphenyl)-4-yl]carbonyl}-5-[(1,3-dihydro-1,3-dioxo-2H-isoindol-2-yl)methyl]-cyclopentane        carboxylic acid inhibited the trafficking of murine peritoneal        macro-phages across this artificial basement membrane (IC₅₀≦1        μM).        LPS Induced TNFα Production in Mice

The in vivo inhibitory properties of selected compounds can bedetermined using a murine LPS induced TNFα production in vivo model.BALB/c mice (Charles River Breeding Laboratories; Kingston, N.Y.) ingroups of ten are treated with either vehicle or compound. After onehour, endotoxin (E. coli lipopolysaccharide (LPS) 100 mg) isadministered intraperitoneally (i.p.). After 90 min, animals areeuthanized by carbon dioxide asphyxiation and plasma is obtained fromindividual animals by cardiac puncture into heparinized tubes, Thesamples are clarified by centrifugation at 12,500×g for 5 min at 4° C.The supernatants are decanted to new tubes, which are stored as neededat −20° C. TNFα levels in sera are measured using a commercial murineTNF ELISA kit (Genzyme).

Other embodiments of the invention will be apparent to the skilled inthe art from a consideration of this specification or practice of theinvention disclosed herein. It is intended that the specification andexamples be considered as exemplary only, with the true scope and spiritof the invention being indicated by the following claims.

Abbreviations:

-   -   DMF: N,N-Dimethylformamide    -   RT: room temperature    -   THF: Tetrahydrofuran

PREPARATION EXAMPLES Example 12-[2-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl]-4-(4′-ethoxy[1,1′-biphenyl]-4-yl)-4-oxobutanoicacid

Intermediate 1A

4-Ethoxy-1,1′-biphenyl

Iodoethane (68.7 g, 35,57 mL, 440.6 mmol) was added to a suspension of50 g (170.2 mmol) of 4-hydroxy-1,1′-biphenyl and 40.6 g (293.75 mmol)K₂CO₃ in 600 mL acetone. The resulting reaction mixture was stirredunder reflux for 16 hours. After cooling to room temperature the acetonewas removed under reduced pressure, the residue was dissolved in ethylacetate and extracted with water. The aqueous layers where extracted 3times with ethyl acetate, the combined organic phases dried (Na₂SO₄) andevaporated to yield 56 g of the desired compound as a colorless solid.

Yield: 56 g (96%).

¹H-NMR (d₆-DMSO): 7.55-7.65 (m, 4H), 7.42 (t, J=8 Hz, 2H), 7.3 (t, J=8Hz, 1H), 6.95-7.05 (m, 2H), 4.07 (q, J=7 Hz, 2H), 1.35 (t, J=7 Hz, 3H).

Intermediate 1B

2-Bromo-1-(4′-ethoxy[1,1′-biphenyl]-4-yl)-1-ethanone

A solution of 56 g (282 mmol) of Intermediate 1A in 1.5 L CH₂Cl₂ wascooled to 0° C. and placed under argon. Bromoacetyl bromide (85.5 g,36,8 mL, 423 mmol) was added, and then AlCl₃ (37.41 g, 280.53 mmol) wasadded in portions over 60 min. After the addition was complete, themixture was stirred for 20 h, warming to RT. The mixture was then pouredslowly into a stirred mixture of 2 kg ice/500 ml conc. HCl. The organiclayer was separated, washed with 2N HCl and water, dried (Na₂SO₄) andevaporated. The crude product was purified by recrystallization(acetonitril) to give 49.3 g (54%) white solid.

¹H-NMR (d₆-DMSO): 8.08 (d, J=8 Hz, 2H), 7.81 (d, J=8 Hz, 2H), 7.73 (d,J=8 Hz, 2 Hz), 7.06 (d, J=8 Hz, 2H), 4.95 (s, 2H), 4.1 (q, J=7 Hz, 2H),1:36 (t, J=7 Hz, 3H).

Intermediate 1C

Di(tert-butyl)2-[2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl]-2-[2-(4′-ethoxy-1,1′-biphenyl]-4-yl)-2-oxoethyl]malonate

A solution of Intermediate 5F (3.2 g, 10 mmol) in 50 mL DMF was addeddropwise to a suspension of NaH (500 mg, 12.5 mmol) in 20 mL DMF andstirred for 30 min at RT. Intermediate 1B (3.9 g, 10 mmol) in 30 ml DMFwas added slowly and the resulting mixture was stirred for 4 h at RT.The reaction was quenched with saturated NH₄Cl solution, extracted twicewith diethyl ether, washed with saturated NaHCO₃, water and brine, dried(Na₂SO₄) and evaporated. The crude product was purified using flashchromatography (hexane/ethyl acetate: 1/1).

Yield: 4.96 g (71%).

¹H-NMR (d₆-DMSO): 8.02 (d, J=8 Hz, 2H), 7.68-7.81 (m, 8H), 7.05 (d, J=8Hz, 2H), 4.1 (q, J=7 Hz, 2H), 3.72 (s, 2H), 3.55-3.65 (m, 2H), 2.26-2.87(m, 2H), 1.33-1.4 (m, 18H).

Example 12-[2-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl]-4-(4′-ethoxy[1,1′-biphenyl]-4-yl)-4-oxobutanoicacid

2.2 g (3.51 mmol) of Intermediate 1C was added in one portion to acooled (0° C.) 1:1 mixture of CH₂Cl₂ and trifluoroacetic acid. Thereaction mixture was stirred overnight at RT, evaporated and dried undervacuum. The residue was dissolved in 5 mL dioxane and heated for 5 hunder reflux. The rection mixture was evaporated, the residue trituratedwith diethyl ether, stirred for 15 min and filtered. The remaining solidwas dried under vacuum.

Yield: 1.21 g (73%).

¹H-NMR (d₆-DMSO): 12.28 (s, 1H), 8.02 (d, J=8 Hz, 2H), 7.67-7.9 (m, 8H),7.05 (d, I=8 Hz, 2H), 4.1 (q, J=7 Hz, 2H), 3.72 (t, J=7.5 Hz, 2H), 3.49(dd, J=17.5 Hz, J=8 Hz, 2H), 3.28 (dd, J=17.5 Hz, J=5 Hz, 2H), 2.78-2.95(m, 1H), 1.76-2.13 (m, 2H), 1.38 (t, J=7 Hz, 3H).

The racemate of Example 1 was separated into its pure enantiomers viachiral HPLC using a commercially available 5 μm Kromasil KR100-5-CHI-DMB phase. A solvent mixture consisting of 50% iso-hexane and50% of a tert-butylmethyl ether/dichloromethane/glacial acetic acidmixture (480:40:1) was employed at a constant flow rate of 25 ml/min.

Example 2(+)-2-[2-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl]-4-(4′-ethoxy[1,1′-biphenyl]-4-yl)-4-oxobutanoicacid

Faster eluting enantiomer:

Yield: 374 mg (42%).

[α]²³ _(D)+6.33° (c=0.47 in THF).

Example 3(−)-2-[2-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl]-4-(4′-ethoxy[1,1′-biphenyl]-4-yl)-4-oxobutanoicacid

Slower eluting enantiomer:

Yield: 321 mg (36%).

[α]²³ _(D)−5.6° (c=0.5 in THF).

Example 4

-   -   (+)-4-(4′-Chloro[1,1′-biphenyl]4-yl)-2-[2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl]-4-oxobutanoic        acid

The compound of Example 4 was prepared according to the procedure givenfor Example 268 in WO 96/15096.

[α]²³ _(D)+5.55° (c=0.525 in THF).

Example 5(rac)-4-[4′-(Acetyloxy)[1,1′-biphenyl]-4-yl]-2-[2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)-ethyl]-4-oxobutanoicacid

Intermediate 5A

4′-(2-Bromoacetyl)[1,1′-biphenyl]-4-yl acetate

A solution of 50 g (236 mmol) of [1,1′-biphenyl]-4-yl acetate in 500 mldichloromethane was placed under argon and cooled to 0° C. Bromoacetylbromide (31.6 ml, 363 mmol) was added, followed by aluminium chloride(94.3 g, 707 mmol) which was added in portions under vigorous stirringover 30 min. The resulting mixture was stirred at 0° C. for a further 30min and at room temperature overnight. The mixture was then slowlypoured into 500 ml of cold 10% HCl and extracted three times withdichloromethane. The organic layer was dried over Na₂SO₄, filtered andevaporated. The residue was triturated with 1:1 diisopropylether/isopropanol, filtered, and the remaining solid dried under vacuum.

Yield: 73.3 g (93.4%).

¹H-NMR (CDCl₃): δ=2.34 (s, 3H), 4.48 (s, 2H), 7.21 (m, 2H), 7.66 (m,4H), 8.08 (m, 2H).

Intermediate 5B

2-(Benzyloxy)-1-ethanol

Ethylene glycol (742.5 g, 11.96 mol) was added to a solution of sodiumhydroxide pellets (475.2 g, 11.88 mol) in 450 ml of water kept at 80° C.Benzyl chloride (302.8 g, 2.39 mol) was then added at 65° C., and theresulting suspension was vigorously stirred at 120° C. overnight. Aftercooling to room temperature, the mixture was poured into ice-water andextracted five times with diethyl ether. The combined organic layerswere washed with brine, dried over Na₂SO₄, filtered and evaporated. Theremaining residue was then distilled under vacuum and the relevantfractions (bp. 95-125° C. at 0.1-1 mbar) collected.

Yield: 175 g (48.1%) of a colourless liquid.

¹H-NMR (CDCl₃): δ=2.09 (tr, 1H), 3.60 (m, 2H), 3.77 (m, 2H); 4.56 (s,2H) 7.35 (m, 5H).

Intermediate 5C

Benzyl 2-chloroethyl ether

Thionyl chloride (41.2 ml, 567.6 mmol) was slowly added to a mixture ofIntermediate 5B (90 g, 80% purity, 473.1 mmol) and N,N-dimethyl aniline(76.5 ml, 597.5 mmol) while keeping the reaction temperature at 50° C.by ice-water cooling. After stirring at 50° C. for 1 h, further portionsof N,N-dimethyl aniline (15.3 ml, 119.5 mmol) and thionyl chloride (8.2ml, 113.5 mmol) were added, and the mixture was stirred at 50° C. foranother 2 h and at room temperature overnight. The solution was thenpoured into a mixture of ice-water (200 ml) and conc. HCl (100 ml) andextracted three times with dichloromethane. The combined organic layerswere washed twice with 10% HCl and twice with water, dried over Na₂SO₄,filtered and evaporated. The remaining residue was then distilled undervacuum (water pump) and the relevant fractions collected.

Yield: 69.1 g (85.6%) of a colourless liquid.

¹H-NMR (CDCl₃): δ=3.69 (m, 4H), 4.59 (s, 2H), 7.35 (m, 5H).

Intermediate 5D

Di(tert-butyl) 2-[2-(benzyloxy)ethyl]malonate

Di(tert-butyl) malonate (151.4 g, 686 mmol) was added dropwise at 50° C.to a suspension of potassium tert-butylate (77 g, 686 mmol) in 500 ml oftert-butanol. Sodium iodide (10.33 g) was then added, followed bydropwise addition of Intermediate 5C (117.1 g, 686 mmol) at 40-50° C.The resulting thick suspension was stirred at 70° C. for two days.During this time, two further portions of potassium tert-butylate (15.4g each, 70 mmol) were added. The mixture was then poured into ice-waterand extracted three times with diethyl ether. The organic layers weredried over Na₂SO₄, filtered and evaporated. The crude product wasfinally purified by column chromatography using a cyclohexane/ethylacetate gradient (70:1→15:1).

Yield: 134 g (55.8%) of a colourless oil.

¹H-NMR (DMSO-d₆): δ=1.38 (s, 18H), 1.96 (q, 2H), 3.31 (tr, 1H), 3.41(tr, 2H), 4.43 (s, 2H), 7.31 (m, 5H).

Intermediate 5E

Di(tert-butyl) 2-(2-hydroxyethyl)malonate

A solution of Intermediate 5D (46.58 g, 132.9 mmol) in 300 ml ethanolwas hydrogenated at atmospheric pressure in the presence of 10%palladium on charcoal (2.0 g). After stirring for 3 h at roomtemperature, another 1.0 g portion of palladium catalyst was added, andstirring was continued at room temperature overnight. The mixture wasthen filtered through celite, evaporated, and the crude product purifiedby column chromatography using a dichloromethane/methanol gradient(70:1→30:1).

Yield; 23.3 g (67.2%) of a pale yellow oil.

¹H-NMR (CDCl₃): δ=1.47 (s, 18H), 1.96 (tr, 1H), 2.08 (q, 2H), 3.36 (tr,1H), 3.72 (q, 1H).

Intermediate 5F

Di(tert-butyl)2-[2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl]malonate

To a stirred solution of Intermediate 5E (30.0 g, 115.2 mmol) in 255 mlof dry THF were added successively phthalimide (21.4 g, 144.1 mmol),triphenyl phosphine (35.1 g, 132.5 mmol) and, at 0° C., diethylazodicarboxylate (22.1 g, 126.8 mmol). The resulting solution wasstirred overnight while warming up to room temperature, then dilutedwith ethyl acetate and washed twice with water and with brine. Theorganic phase was dried over Na₂SO₄, filtered and evaporated. The crudeproduct was finally purified by column chromatography using acyclohexane/dichloromethane/ethyl acetate gradient (7:1:1→5:1:1).

Yield: 10.02 g (22.3%) of a white solid.

¹H-NMR (DMSO-d₆): δ=1.37 (s, 18H), 2.03 (q, 2H), 3.30 (tr, 1H), 3.63(tr, 2H), 7.85 (m, 4H).

Intermediate 5G

Di(tert-butyl)2-{2-[4′-(acetyloxy)[1,1′-biphenyl]-4-yl]-2-oxoethyl}-2-[2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl]malonate

Under argon, a solution of Intermediate 5F (6.5 g, 16.69 mmol) in 60 mlof dry THF was added dropwise at 0° C. to a suspension of sodium hydride(0.51 g, 80% suspension in mineral oil, 16.86 mmol) in 30 ml of dry THF.After stirring at 30-40° C. for 30 min, the mixture was recooled to 0°C., and a solution of Intermediate 5A (5.6 g, 16.86 mmol) in 60 ml ofdry THF was added dropwise. The mixture was then stirred overnight whilewarming up to room temperature. Further portions of sodium hydride (0.1g, 3.4 mmol) and Intermediate 5A (1.12 g, 3.4 mmol) were added at 0° C.,and stirring was continued at room temperature for another 3 h. Thereaction mixture was quenched by addition of saturated ammonium chloridesolution (100 ml) and brine (200 ml), and extracted twice with ethylacetate. The organic phase was dried over Na₂SO₄, filtered andevaporated. The crude product was finally purified by columnchromatography using a dichloro-methane/ethyl acetate gradient(50:1→30:1).

Yield: 4.41 g (41.2%) of an off-white solid.

¹H-NMR (DMSO-d₆): δ=1.38 (s, 18H), 2.32 (m, 5H), 3.61 (tr, 2H), 3.73 (s,2H), 7.28 (d, 2H), 7.81 (m, 8H), 8.04 (d, 2H).

Example 5(rac)-4-[4′-(Acetyloxy)[1,1′-biphenyl]-4-yl]-2-[2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl]-4-oxobutanoicacid

Intermediate 5G (400 mg, 0.62 mmol) was dissolved at 0° C. in a mixtureof dichloromethane (5 ml) and trifluoroacetic acid (5 ml). Afterstirring at room temperature for 1.5 h, 10 ml of toluene were added, andthe reaction mixture was evaporated. The residue was dried under vacuum,then re-dissolved in 20 ml of dioxane, and the solution heated underreflux for 6 h. The mixture was evaporated to dryness, the residuetriturated with diethyl ether, filtered, and the remaining solid driedunder vacuum to give the final product

Yield: 261 mg (86.2%) of an off-white solid.

¹H-NMR (DMSO-d₆): δ=1.95 (m, 2H), 2.31 (s, 3H), 2.89 (m, 1H), 3.38 (m,2H), 3.72 (tr, 2H), 7.28 (d, 2H), 7.84 (m, 8H), 8.07 (d, 2H), 12.33 (brs, 1H).

Example 6(rac)-2-[2-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl]-4-(4′-hydroxy[1,1′-biphenyl]-4-yl)-4-oxobutanoicacid

Intermediate 6A

Di(tert-butyl) 2-[2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl]-2-[2-(4′-hydroxy-[1,1′-biphenyl]-4-yl)-2-oxoethyl]malonate

Finely powdered, anhydrous potassium carbonate (2.15 g, 15.58 mmol) wasadded to a solution of Intermediate 5G (2.0 g, 3.12 mmol) in 90 ml of aTHF/methanol/ethanol mixture (30:50:10). The resulting suspension wasvigorously stirred at room temperature for 45 min, then diluted withethyl acetate and filtered. The filtrate was concentrated under vacuumto half of its original volume, diluted again with ethyl acetate, andthen poured into an ice-cold pH 4 buffer solution. The aqueous phase wasextracted twice with ethyl acetate, and the combined organic layers weredried over Na₂SO₄, filtered and evaporated. After drying under vacuum,700 mg (approx. 1.1 mmol) of the residue (which contained somering-opened material) were dissolved in 20 ml of dichloromethane, and1-hydroxy-1H-benzotriazol hydrate (189 mg, 1.23 mmol) andN′-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (229 mg,1.18 mmol) were added at 0° C. The reaction mixture was stirred at roomtemperature for 3 days, then diluted with dichloromethane, and washedtwice with pH 4 buffer solution and with saturated sodiumhydrogencarbonate solution. The organic phase was dried over Na₂SO₄,filtered and evaporated. The crude product was finally purified bycolumn chromatography using a dichloromethane/methanol gradient(100:1→80:1).

Yield: 494 mg (71%) of a white solid.

¹H-NMR (DMSO-d₆): δ=1.38 (s, 18H), 2.31 (m, 2H), 3.60 (m, 2H), 3.70 (s,2H), 6.90 (d, 2H), 7.61 (d, 2H), 7.78 (m, 6H), 8.00 (d, 2H), 9.76 (s,1H).

Example 6(rac)-2-[2-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl]-4-(4′-hydroxy[1,1′-biphenyl]-4-yl)-4-oxobutanoicacid

Intermediate 6A (490 mg, 0.82 mmol) was dissolved at 0° C. in a mixtureof dichloromethane (7.5 ml) and trifluoroacetic acid (7.5 ml). Afterstirring at room temperature for 30 min, 7 ml of toluene were added, andthe reaction mixture was evaporated. The residue was dried under vacuum,then re-dissolved in 15 ml of dioxane, and the solution heated underreflux for 4.5 h. After cooling to room temperature, diethyl ether (15ml) was added to the reaction mixture, and the precipitated productcollected by filtration. The filtrate was evaporated to dryness, theresidue triturated with diethyl ether, containing a few drops ofmethanol, and filtered again to give a second crop of the final product.

Yield: 299 mg (82.6%) of a white solid.

¹H-NMR (DMSO-d₆): δ=1.94 (m, 2H), 2.87 (m, 1H), 3.38 (m, 2H), 3.70 (tr,2H), 6.89 (d, 2H), 7.61 (d, 2H), 7.75 (d, 2H), 7.85 (m, 4H), 8.01 (d,2H), 9.76 (br s, 1H), 12.30 (br s, 1H).

Example 7(rac)-4-(4′-Chloro[1,1′-biphenyl]-4-yl)-2-[2-(4,6-dimethoxy-1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl]-4-oxobutanoicacid

Intermediate 7A

2-Bromo-1-(4′-chloro[1,1′-biphenyl]-4-yl)-ethanone

This intermediate was prepared as described in the indicated referenceWO 96/15096.

Intermediate 7D

Di(tert-butyl)2-[2-(4,6-dimethoxy-1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl]malonate

To a stirred solution of Intermediate 5E (8.4 g, 32.2 mmol) in 100 ml ofdry THF were added successively 3,5-diethoxyphthalimide (10.0 g, 48.3mmol), triphenyl phosphine (11.1 g, 41.8 mmol) and, at 0° C., diethylazodicarboxylate (6.7 g, 38.6 mmol). The resulting solution was stirredovernight while warming up to room temperature. After filtration, thefiltrate was diluted with ethyl acetate and washed twice with water andwith brine. The organic phase was dried over Na₂SO₄, filtered andevaporated. The crude product was finally purified by columnchromatography using a dichloromethane/ethyl acetate gradient(70:1→30:1).

Yield: 2.69 g (18.6%) of a white solid.

¹H-NMR (DMSO-d₆): δ=1.38 (s, 18H), 1.97 (q, 2H), 3.23 (tr, 1H), 3.54(tr, 2H), 3.92 (s, 6H), 6.89 (d, 1H), 6.97 (d, 1H).

Intermediate 7C

Di(tert-butyl)2-[2-(4′-chloro[1,1′-biphenyl]-4-yl)-2-oxoethyl]-2-[2-(4,6-dimethoxy-1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl]malonate

Under argon, a solution of Intermediate 7B (2.69 g, 5.98 mmol) in 30 mlof dry THF was added dropwise at 0° C. to a suspension of sodium hydride(0.18 g, 80% suspension in mineral oil, 6.04 mmol) in 20 ml of dry THF.After stirring at 30-40° C. for 30 min, the mixture was re-cooled to 0°C., and a solution of Intermediate 3A (1.87 g, 6.04 mmol) in 20 ml ofdry THF was added dropwise. The mixture was then stirred overnight whilewarming up to room temperature. Further portions of sodium hydride (36mg, 1.2 mmol) and Intermediate 7A (374 mg, 1.2 mmol) were added at 0°C., and stirring was continued at room temperature for three days. Thereaction mixture was quenched by addition of saturated ammonium chloridesolution (40 ml) and brine (80 ml), and extracted twice with ethylacetate. The organic phase was dried over Na₂SO₄, filtered andevaporated. The crude product was finally purified by columnchromatography using a dichloro-methane to dichloromethane/ethyl acetate(20:1) gradient.

Yield: 1.51 g (37.2%) of a white solid.

R_(F)=0.51 (dichloromethane/ethyl acetate 20:1);

-   -   =0.59 (cyclohexane/ethyl acetate 1:1).

ESI-MS: m/z=678 [M+H]⁺, 622 ([M+H]⁺−C₄H₈), 566 ([M+H]⁺−2×C₄H₈), 548([M+H]⁺−2×C₄H₈/−H₂O).

Example 74-(4′-Chloro[1,1′-biphenyl]-4-yl)-2-[2-(4,6-dimethoxy-1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl]oxobutanoicacid

Intermediate 7C (1.5 g, 2.2 mmol) was dissolved at 0° C. in a mixture ofdichloromethane (10 ml) and trifluoroacetic acid (10 ml). After stirringat room temperature for 45 min, 10 ml of toluene were added, and thereaction mixture was evaporated.

The residue was dried under vacuum, then redissolved in 20 ml ofdioxane, and the solution heated under reflux for 6 h. The mixture wasevaporated to dryness, the residue triturated with diethyl ether,filtered, and the remaining solid dried under vacuum to give the finalproduct.

Yield: 1.07 g (92.1%) of an off-white solid.

¹H-NMR (DMSO-d₆): δ=1.89 (m, 2H), 2.84 (m, 1H), 3.39 (m, 2H), 3.63 (tr,2H), 3.92 (s, 6H), 6.88 (d, 1H), 6.97 (d, 1H), 7.5w (d, 2H), 7.82 (m,4H), 8.07 (d, 2H), 12.30 (br s, 1H).

Examples 8 and 9 (+)- and(−)-4-(4′-Bromo[1,1′-biphenyl]-yl)-2-[2-(1,3-dioxo-1,3-dihydro-2H-isoindol2-yl)ethyl]-4-oxobutanoicacid

Racemic4-(4′-Bromo[1,1′-biphenyl]-4-yl)-2-[2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)-ethyl]-4-oxobutanoicacid was prepared essentially as described in the indicated reference WO96/15096.

¹H-NMR (DMSO-d₆): δ=1.95 (m, 2H), 2.88 (m, 1H), 3.38 (m, 2H), 3.72 (tr,2H), 7.72 (m, 4H), 7.85 (m, 6H), 8.08 (d, 2H), 12.33 (br s, 1H).

1.0 g (1.97 mmol) of this material was separated into pure enantiomersby chiral HPLC using a commercially available 5 μm Kromasil KR100-5-CHI-DMB phase. A solvent mixture consisting of 40% iso-hexane and60% of a tert-butylmethyl ether/dichloromethane/glacial acetic acidmixture (480:40:1) was employed at a constant flow rate of 25 ml/min.

Example 8

First eluting enantiomer A:

Yield: 309 mg (31%).

[α]_(D) ²⁰=+3.87° (c=0.458 g/100 ml, THF).

Example 9

Second eluting enantiomer B:

Yield: 240 mg (24%).

[α]_(D) ²⁰=−5.98° (c=0.482 g/100 ml, THF).

Examples 10 and 11 (+)- and(−)-4-(4′-Chloro[1,1′-bipenyl]-4-yl)-2-[2-(5,7-dioxo-5,7-dihydro-6H-[1,3]-dioxolo[4,5-f]isoindol-6-yl)ethyl]-4-oxobutanoicacid

Racemic4-(4′-Chloro[1,1′-bipenyl]-4-yl)-2-[2-(5,7-dioxo-5,7-dihydro-6H-[1,3]-dioxolo[4,5-f]isoindol-6-yl)ethyl]-4-oxobutanoicacid was prepared essentially as described in the indicated reference WO96/15096.

¹H-NMR (DMSO-d₆): δ=1.91 (m, 2H), 2.85 (m, 1H), 3.38 (m, 2H), 3.66 (tr,2H), 6.26 (s, 2H), 7.39 (s, 2H), 7.58 (d, 2H), 7.82 (m, 4H), 8.06 (d,2H), 12.31 (br s, 1H).

0.70 g (1.38 mmol) of this material was separated into pure enantiomersby chiral HPLC using a commercially available 1 μm Kromasil KR100-5-CHI-MDB phase. A solvent mixture consisting of 40% isohexane and60% of a tert-butylmethyl ether/dichloromethane/glacial acetic acidmixture (480:40:1) was employed at a constant flow rate of 25 ml/min.

Example 10

First eluting enantiomer A:

Yield: 261 mg (37.3%).

[α]_(D) ²⁰=+13.99° (c=0.955 g/100 ml, THF).

Example 11

Second eluting enantiomer B:

Yield: 228 mg (32.6%).

[α]_(D) ²⁰=−15.15° (c=0.991 g/100 ml, THF).

Example 124-(4′-Cyano[1,1′-biphenyl]-4-yl)-4-oxo-2-{2-[4-oxo-1,2,3-benzotriazin-3(4H)-yl]ethyl}butanoicacid

Intermediate 12A

4′-(2-Bromoacetyl)[1,1′-biphenyl]-4-carbonitrile

4.68 g aluminium chloride (35.15 mmol) are dissolved in 45 mldichloromethane and treated dropwise with 3.38 g (16.74 mmol)bromoacetyl bromide at 0° C. After 30 min 3 g (16.74 mmol)4-cyanobiphenyl, dissolved in 15 ml dichloromethane, are added dropwise.The reaction mixture is stirred overnight at ambient temperature, addedto ice-water and extracted 2 times with dichloromethane. The organicphase is washed with water and brine, dried and evaporated. The residueis triturated with petrol ether, filtered and dried. Yield: 4.24 g(83%).

200 MHz ¹H-NMR (CDCl₃): 4.49, s, 2H; 7.73, m, 6H; 8.11, d, 2H.

Intermediate 12B

Di(tert-butyl)-2-[2-(4′-cyano[1,1′-biphenyl]-4-yl)-2-oxoethyl]-2-(2-[4-oxo-1,2,3-benzo-triazin-3(4H)-yl]ethyl)malonate

A solution of 2.59 g (6.67 mmol) of Intermediate 13C in 20 ml DMF isadded dropwise to a suspension of 0.333 g NaH (60% in mineral oil) in 20ml DMF. The mixture is stirred for 30 min and a solution of 2 g (6.67mmol) of Intermediate 12A in 20 ml DMF is added. The mixture is stirredfor 2.5 h at RT, poured onto 150 ml NH₄Cl solution and extracted withethyl acetate. The organic phase is washed with water and brine, driedover MgSO₄ and evaporated. The residue is purified by chromatography togive 0.62 g (15%).

200 MHz ¹H-NMR (CDCl₃): 2.71, s, 2H; 3.81, s, 2H; 4.53, m, 2H; 7.75, m,7H; 7.91, m, 1H; 8.10, m, 3H; 8.30, m, 1H.

Example 124-(4′-Cyano[1,1′-biphenyl]-4-yl)-4-oxo-2-{2-[4-oxo-1,2,3-benzotriazin-3(4H)-yl]-ethyl}butanoicacid

0.61 g (1 mmol) of Intermediate 12B is dissolved in 10 mldichloro-methane/trifluoroacetic acid (1:1) at 0° C. and the mixture isstirred for 2 h at RT. After adding toluene the reaction mixture isevaporated to dryness and the residue taken into 10 ml dioxane. Thesolution is stirred under reflux for 6 h and at RT overnight. Thesolvent is removed in vacuo and the residue purified by HPLC to yield 96mg (21%).

200 MHz ¹H-NMR (CDCl₃): 2.28, m, 2H; 2.49, m, 2H; 3.26, m, 1H; 4.58, m,2H, 7.72, m, 10H; 8.18, m, 1H; 8.38, m, 1H.

Example 134-Oxo-2-{2-[4-oxo-1,2,3-benzotriazin-3(4H)-yl]ethyl}-4-[4′-(trifluoromethoxy)[1,1′-biphenyl]-4-yl]butanoicacid

Intermediate 13A

1-[4-(Trifluoromethoxy)[1,1′-biphenyl]-4-yl-]-1-ethanone

To a mixture of 30 g (126 mmol) 4-(trifluoromethoxy)-1,1′-biphenyl and21 g aluminium trichloride (157 mmol) in 120 ml nitrobenzene are addedat a temperature below 20° C. 9.87 g (126 mmol) acetyl chloride. Thereaction mixture is stirred for 2 h at 0° C., added to 240 ml ice-waterand 42 ml conc. HCl and extracted with ethyl acetate. The organic phaseis washed with water and brine and the solvents removed in vacuo. Theresidue is triturated with petrol ether, filtrated and dried. From thefiltrate another batch can be obtained after crystallizing at 4° C. togive overall 23.5 g (66%).

200 MHz ¹H-NMR (CDCl₃): 2.65, s, 3H; 7.33, d, 2H; 7.58, m, 4H; 8.06, d,2H.

Intermediate 13B

2-Bromo-1-[4′-(trifluoromethoxy)[1,1′-biphenyl]-4-yl]-1-ethanone

12.27 g (43.8 mmol) of Intermediate 13A are dissolved in a mixture of150 ml methanol, 150 ml ethanol and 50 ml ether with gentle heating.5.914 g (56.9 mmol) boronic acid trimethylester are added at RT and 7.35g (45.9 mmol) bromine are added dropwise. The reaction mixture isstirred until disappearance of the red-brown colour. Solvents areremoved in vacuo and the residue is purified by chromatography(cyclohexane/ethyl acetate) to give 6.2 g (39%).

200 MHz ¹H-NMR (CDCl₃): 4.49, s, 2H; 7.33, d, 2H; 7.68, dd, 4H; 8.10, d,2H.

Intermediate 13C

Di(tert-butyl) 2-{2-[4-oxo-1,2,3-benzotriazin-3(4H)-yl]ethyl}malonate

46.2 g (177 mmol) of Intermediate 5E are dissolved in 600 ml THF. 69.8 g(266 mmol) triphenylphosphin und 39.2 g (266 mmol)1,2,3-benzotriazin-4(3H)-one are added. 46.4 g (266 mmol) DEAD are addeddropwise. The reaction mixture was stirred overnight at roomtemperature.The solvent is removed in vacuo and the product obtained bychromatography (cyclohexan/ethylacetate 6:1).

Yield: 51.8 g (63%).

200 MHz ¹H-NMR (CDCl₃): 1.43, s, 18H; 2.43, quar., 2H; 3.30, t, 1H;4.57, t, 2H; 7.80, m, 1H; 7.94, m, 1H; 8.16, dd, 1H; 8.36, dd, 1H.

Intermediate 13D

Di(tert-butyl)-2-{2-[4-oxo-1,2,3-benzotriazin-3(4H)-yl]ethyl}-2-{2-oxo-2-[4′-(trifluoromethoxy)[1,1′-biphenyl]-4-yl]ethyl}malonate

To a suspension of 0.52 g (12.9 mmol) sodium hydride (60% suspension inmineral oil) in 20 ml DMF is added a solution of 4.04 g (10.37 mmol) ofIntermediate 13C in 30 ml DMF dropwise. After stirring for 30 min at RTa solution of 3.73 g (10.37 mmol) of Intermediate 13B in 30 ml DMF isadded dropwise and the reaction mixture is stirred for 2 h at RT. Thereaction mixture is poured onto NH₄Cl solution, extracted with ethylacetate and the organic phase is washed with water and brine. Afterdrying and evaporation of the solvents the product is purified bychromatography to give 2.09 g (30%).

200 MHz ¹H-NMR (CDCl₃): 2.70, m, 2H; 3.82, s, 2H; 4.54, m, 2H; 7.32, d,2H; 7.62, m, 5H; 8.07,m, 4H; 8.30, m, 1H.

Example 134-Oxo-2-{2-[4-oxo-1,2,3-benzotriazin-3(4H)-yl]ethyl}-4-[4′-(trifluoromethoxy)[1,1′-biphenyl]-4-yl]butanoicacid

A solution of 2.09 g (3.13 mmol) of Intermediate 13C in 15 mldichloromethane and 15 ml trifluoroacetic acid is stirred at RT for 2 h.After adding toluene the solvents are removed in vacuo. The residue isdissolved in 30 ml dioxane, the solution is refluxed for 6 h and stirredat RT overnight. Solvents are removed in vacuo, the residue istriturated with ether, the precipitate is collected by filtration anddried to give 0.51 g (32%).

200 MHz ¹H-NMR (DMSO-d₆): 2.18, m, 2H; 2.98, m, 1H; 3.51, m, 2H; 4.52,m, 2H; 7.52, d, 2H; 7.89, m, 5H; 8.10, m, 3H; 8.24, m, 2H; 12.39, s, 1H.

Examples 14 and 15 (+)- and(−)-(1S*,2S*,5R*)-2-[(4′-Chloro[1,1′-biphenyl]-4-yl)carbonyl]-5-[(1,3-dioxo1,3-dihydro-2H-isoindol-2-yl)methyl]cyclopentanecarboxylicacid

The racemic compound was prepared essentially following the procedurefor Example 360 of WO 96/15096.

4.20 g (8.61 mmol) of this material was separated into pure enantiomersby chiral HPLC using a commercially available 5 μm Kromasil KR100-5-CHI-MDB phase A solvent mixture consisting of 30% iso-hexane and70% of a tert-butylmethyl ether/dichloromethane/glacial acetic acidmixture (480:40:1) was employed at a constant flow rate of 25 ml/min.

Example 14

First eluting enantiomer A:

Yield: 1.72 g (41.0%).

[α]_(D) ²¹=+44.26° (c=0.464 g/100 ml, THF).

Example 15

Second eluting enantiomer B:

Yield: 1.59 g (37.9%).

[α]_(D) ²¹=−43.70° (c=0.575 g/100 ml, THF).

1. Compounds of the general formula (I′)

wherein CO—E—CO₂H represents a 3-carboxyl-5-R⁷-pentan-1-on-1-yl-residueand the substituents T and R⁷ have the meaning indicated in thefollowing table: racemate, (+)- or (−)- T R⁷ enantiomer OAc

rac ; OH

rac ; Cl

rac ; CN

rac or OCF₃

rac .


2. A pharmaceutical composition comprising a compound according to claim1 and a pharmaceutically acceptable carrier.
 3. A method of treatingacute and chronic inflammatory processes, comprising administering to amammal an effective amount of a compound according to claim
 1. 4. Amethod of treating a respiratory disease, comprising administering to amammal an effective amount of a compound having matrix metalloproteaseinhibitory activity and the generalized formula:(T)_(x)A—B—D—E—CO₂H wherein (a) (T)_(x)A represents;

wherein each T represents a substituent group, independently selectedfrom the group consisting of: the halogens —F, —Cl, —Br, and —I; alkylof 1-10 carbons; haloalkyl of 1-10 carbons; haloalkoxy of 1-10 carbons;alkenyl of 2-10 carbons; alkynyl of 2-10 carbons; —(CH₂)_(p)Q, wherein p is 0 or an integer 1-4, -alkenyl-Q, wherein said alkenyl moietycomprises 2-4 carbons, and -alkynyl-Q, wherein said alkynyl moietycomprises 2-7 carbons; and Q is selected from the group consisting ofaryl of 6-10 carbons, heteroaryl comprising 4-9 carbons and at least oneN, O, or S heteroatom, —CN, —CHO, —NO₂, —CO₂R², —OCOR², —SOR³, —SO₂R³,—CON(R⁴)₂, —SO₂N(R⁴)₂, —C(O)R², —N(R⁴)₂, —N(R²)COR², —N(R²)CO₂R³,—N(R²)CON(R⁴)₂, —CHN₄, —OR⁴, and —SR⁴; wherein R² represents H;  alkylof 1-6 carbons;  aryl of 6-10 carbons;  heteroaryl comprising 4-9carbons and at least one N, O, or S heteroatom; or  arylalkyl in whichthe aryl portion contains 6-10 carbons and the alkyl portion contains1-4 carbons; or  heteroaryl-alkyl in which the heteroaryl portioncomprises 4-9 carbons and at least one N, O, or S heteroatom and thealkyl portion contains 1-4 carbons; R³ represents alkyl of 1-4 carbons; aryl of 6-10 carbons;  heteroaryl comprising 4-9 carbons and at leastone N, O, or S heteroatom; or  arylalkyl in which the aryl portioncontains 6-10 carbons and the alkyl portion contains 1-4 carbons; or heteroaryl-alkyl in which the heteroaryl portion comprises 4-9 carbonsand at least one N, O, or S heteroatom and the alkyl portion contains1-4 carbons; R⁴ represents H;  alkyl of 1-12 carbons;  aryl of 6-10carbons;  heteroaryl comprising 4-9 carbons and at least one N, O, or Sheteroatom;  arylalkyl in which the aryl portion contains 6-10 carbonsand the alkyl portion contains 1-4 carbons;  heteroaryl-alkyl in whichthe heteroaryl portion comprises 4-9 carbons and at least one N, O, or Sheteroatom and the alkyl portion contains 1-4 carbons;  alkenyl of 2-12carbons;  alkynyl of 2-12 carbons;  —(C_(q)H_(2q)O)_(r)R⁵ wherein q is1-3; r is 1-3; and R⁵ is H provided q is greater than 1, or alkyl of 1-4carbons, or phenyl;  alkylenethio terminated with H, alkyl of 1-4Carbons, or phenyl;  alkyleneamino terminated with H, alkyl of 1-4carbons, or phenyl;  —(CH₂)_(s)X wherein s is 1-3 and X is halogen; —C(O)OR²; or  —C(O)R²; and with the provisos that a) when two R⁴ groupsare situated on a nitrogen, they may be joined by a bond to form aheterocycle, and b) unsaturation in a moiety which is attached to Q orwhich is part of Q is separated from any N, O, or S of Q by at least onecarbon atom, and x is 0, 1, or 2; (b) B represents an optionallysubstituted p-phenylene ring containing 0-2 substituents T, whichsubstituents T may independently have the meaning specified under (a);(c) D represents

(d) E represents a chain of n carbon atoms bearing m substituents R⁶,wherein said R⁶ groups are independent substituents, or constitute spiroor nonspiro rings in which a) two groups R⁶ are joined, and takentogether with the chain atom(s) to which said two R⁶ group(s) areattached, and any intervening chain atoms, constitute a 3-7 memberedring, or b) one group R⁶ is joined to the chain on which said one groupR⁶ resides, and taken together with the chain atom(s) to which said R⁶group is attached, and any intervening chain atoms, constitutes a 3-7membered ring; and wherein n is 2 or 3; m is an integer of 1-3; eachgroup R⁶ is independently selected from the group consisting of;fluorine; hydroxyl, with the proviso that a single carbon may bear nomore than one hydroxyl substituent alkyl of 1-10 carbons; aryl of 6-10carbons; heteroaryl comprising 4-9 carbons and at least one N, O, or Sheteroatom; arylalkyl wherein the aryl portion contains 6-10 carbons andthe alkyl portion contains 1-8 carbons; heteroaryl-alkyl wherein theheteroaryl portion comprises 4-9 carbons and at least one N, O, or Sheteroatom, and the alkyl portion contains 1-8 carbons; alkenyl of 2-10carbons; aryl-alkenyl wherein the aryl portion contains 6-10 carbons andthe alkenyl portion contains 2-5 carbons; heteroaryl-alkenyl wherein theheteroaryl portion comprises 4-9 carbons and at least one N, O, or Sheteroatom and the alkenyl portion contains 2-5 carbons; alkynyl of 2-10carbons; aryl-alkynyl wherein the aryl portion contains 6-10 carbons andthe alkynyl portion contains 2-5 carbons; heteroaryl-alkynyl wherein theheteroaryl portion comprises 4-9 carbons and at least one N, O, or Sheteroatom and the alkynyl portion contains 2-5 carbons; —(CH₂)_(t)R⁷wherein t is 0 or an integer of 1-5; and R⁷ is selected from the groupconsisting of

 and corresponding heteroaryl moieties in which the aryl portion of anaryl-containing R⁷ group comprises 4-9 carbons and at least one N, O, orS heteroatom;  wherein Y represents O or S; R¹, R², and R³ are asdefined above; and u is 0, 1, or 2; and —(CH₂)_(v)ZR⁸ wherein v is 0 oran integer of 1 to 4; and Z represents

R⁸ is selected from the group consisting of: alkyl of 1 to 12 carbons;aryl of 6 to 10 carbons; heteroaryl comprising 4-9 carbons and at leastone N, O, or S heteroatom; arylalkyl wherein the aryl portion contains 6to 12 carbons and the alkyl portion contains 1 to 4 carbons;heteroaryl-alkyl wherein the aryl portion comprises 4-9 carbons and atleast one N, O, or S heteroatom and the alkyl portion contains 1-4carbons; —C(O)R⁹ wherein R⁹ represents alkyl of 2-6 carbons, aryl of6-10 carbons, heteroaryl comprising 4-9 carbons and at least one N, O,or S heteroatom, or arylalkyl in which the aryl portion contains 6-10carbons or is heteroaryl comprising 4-9 carbons and at least one N, O,or S heteroatom, and the alkyl portion contains 1-4 carbons; and withthe provisos that  when R⁸ is —C(O)R⁹, Z is S or O;  when Z is O, R⁸ mayalso be —(C_(q)H_(2q)O)_(r)R⁵ wherein q, r, and R⁵ are as defined above;and —(CH₂)_(w)SiR¹⁰ ₃ wherein w is an integer of 1 to 3; and R¹⁰represents alkyl of 1 to 2 carbons; and with the proviso that aryl orheteroaryl portions of any of said T or R⁶ groups optionally may bear upto two substituents selected from the group consisting of—(CH₂)_(y)C(R⁴)(R³)OH, —(CH₂)_(y)OR⁴, —(CH₂)_(y)SR⁴, —(CH₂)_(y)S(O)R⁴,—(CH₂)_(y)S(O)₂R⁴, —(CH₂)_(y)SO₂N(R⁴)₂, —(CH₂)_(y)N(R⁴)₂,—(CH₂)_(y)N(R⁴)COR³, —OC(R⁴)₂O— in which both oxygen atoms are connectedto the aryl ring, —(CH₂)_(y)COR⁴, —(CH₂)_(y)CON(R⁴)₂, —(CH₂)_(y)CO₂R⁴,—(CH₂)_(y)OCOR⁴, -halogen, —CHO, —CF₃, —NO₂, —CN, and —R³, wherein y is0-4; and R³ and R⁴ are defined as above; and any two R⁴ which areattached to one nitrogen may be joined to form a heterocycle; or a anpharmaceutically acceptable salt thereof.
 5. The method of claim 1,wherein (a) (T)_(x)A represents:

wherein each T represents a substituent group, independently selectedfrom the group consisting of: the halogens —F, —Cl, —Br, and —I; alkylof 1-10 carbons; haloalkyl of 1-10 carbons; alkenyl of 2-10 carbons;alkynyl of 2-10 carbons; —(CH₂)_(p)Q, wherein p is 0 or an integer 1-4,-alkenyl-Q, wherein said alkenyl moiety comprises 2-4 carbons, and-alkynyl-Q, wherein said alkynyl moiety comprises 2-7 carbons; and Q isselected from the group consisting of —OR⁴ and —SR⁴; wherein R⁴represents H;  alkyl of 1-12 carbons;  aryl of 6-10 carbons;  heteroarylcomprising 4-9 carbons and at least one N, O, or S heteroatom; arylalkyl in which the aryl portion contains 6-10 carbons and the alkylportion contains 1-4 carbons;  heteroaryl-alkyl in which the heteroarylportion comprises 4-9 carbons and at least one N, O, or S heteroatom andthe alkyl portion contains 1-4 carbons;  —C(O)OR²; or  —C(O)R²; and withthe proviso that unsaturation in a moiety which is attached to Q orwhich is part of Q is separated from any N, O, or S of Q by at least onecarbon atom, and x is 0, 1, or 2; (b) B represents an optionallysubstituted phenyl ring containing 0-2 substituents T, whichsubstituents T may independently have the meaning specified under (a);(c) D represents

(d) E represents a chain of n carbon atoms bearing m substituents R⁶,wherein said R⁶ groups are independent substituents, or constitutenonspiro rings in which two groups R⁶ are joined, and taken togetherwith the chain atom(s) to which said two R⁶ group(s) are attached, andany intervening chain atoms, constitute a 5 or 6-membered ring; andwherein n is 2 or 3; m is an integer of 1 or 2; each group R⁶ isindependently selected from the group consisting of: arylalkyl whereinthe aryl portion contains 6-10 carbons and the alkyl portion contains1-8 carbons; —(CH₂)_(t)R⁷ wherein t is 0 or an integer of 1-5; and R⁷ isselected from the group consisting of

R² is independently selected from the group consisting of: H; aryl of6-10 carbons —(CH₂)_(v)ZR⁸ wherein v is 0 or an integer of 1 to 4; and Zrepresents

R⁸ is selected from the group consisting of: alkyl of 1 to 12 carbons;aryl of 6 to 10 carbons; heteroaryl comprising 4-9 carbons and at leastone N, O, or S heteroatom; arylalkyl wherein the aryl portion contains 6to 12 carbons and the alkyl portion contains 1 to 4 carbons;heteroaryl-alkyl wherein the aryl portion comprises 4-9 carbons and atleast one N, O, or S heteroatom and the alkyl portion contains 1-4carbons; —C(O)R⁹ wherein R⁹ represents alkyl of 2-6 carbons, aryl of6-10 carbons, heteroaryl comprising 4-9 carbons and at least one N, O,or S heteroatom, or arylalkyl in which the aryl portion contains 6-10carbons or is heteroaryl comprising 4-9 carbons and at least one N, O,or S heteroatom, and the alkyl portion contains 1-4 carbons; and withthe provisos that  when R⁸ is —C(O)R⁹, Z is S or O;  when Z is O, R⁸ mayalso be —(C_(q)H_(2q)O)_(r)R⁵ wherein q, r, and R⁵ are as defined above;and —(CH₂)_(w)SiR¹⁰ ₃ wherein w is an integer of 1 to 3; and R¹⁰represents alkyl of 1 to 2 carbons; and with the proviso that aryl orheteroaryl portions of any of said T or R⁶ groups optionally may bear upto two substituents selected from the group consisting of OR⁴, N(R⁴)₂,—OC(R⁴)₂O— in which both oxygen atoms are connected to the aryl ring,CON(R⁴)₂, OCOR⁴, -halogen, —NO₂, and alkyl with up to 6 carbon atomewherein R⁴ is defined as above; or a pharmaceutically acceptable saltthereof.
 6. The method of claim 4 or 5, wherein at least one of theunits T and R⁶ comprises a heteroaromatic ring.
 7. The method of claim 4or 5, wherein in said E unit, n is 2 and m is
 1. 8. The method of claim4 or 5, wherein the compound is selected from the following group:


9. A method of treating a respiratory disease, comprising administeringto a mammal an effective amount of a compound of the general formula(I′)

wherein T is (C₁-C₄)-alkoxy, chloride, bromide, fluoride, acetoxy,hydroxy, cyano, trifluoromethyl or trifluoromethoxy, CO—E—CO₂Hrepresents a 3-carboxyl-5-R⁷-pentan-1-on-1-yl- or a2-carboxyl-3-(R⁷-methyl)-cyclopentan-1-yl)carbonyl-residue, and R⁷represents a group of the formula

or its salt.
 10. A method of treating a respiratory disease, comprisingadministering to a mammal an effective amount of the compound(+)-2-[2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl]4-(4′-ethoxy[1,1′-biphenyl]-4-yl)-4-oxobutanoicacid,


11. A method of treating a respiratory disease, comprising administeringto a mammal an effective amount of the compound(+)-4-(4′-chloro[1,1′-biphenyl]-4-yl)-2-[2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl]-4-oxobutanoicacid


12. The method of claim 4, 9, 10 or 11, wherein said respiratory diseaseis selected from the group consisting of asthma; chronic obstructivepulmonary diseases including chronic bronchitis and emphysema; cysticfibrosis; bronchiectasis; adult respiratory distress syndrome (ARDS);allergic respiratory disease including allergic rhinitis; diseaseslinked to TNF_(α) production including acute pulmonary fibroticdiseases, pulmonary sarcoidosis, silicosis, coal worker'spneumoconiosis, alveolar injury in mammals, such as human, a farm animalor a domestic pet.